register interest

Professor Ray Owens

Research Area: Protein Science and Structural Biology
Technology Exchange: Protein interaction
Scientific Themes: Protein Science & Structural Biology
Keywords: Protein expression, Crystallization and High throughput
Web Links:
Automated construction and screening of protein expression in the OPPf

Automated construction and screening of protein expression in the OPPf

Automated imaging of protein crystallization experiments in the OPPF

Automated imaging of protein crystallization experiments in the OPPF

The Oxford Protein Production Facility-UK (OPPF-UK) is a core facility for protein production at the Research Complex at Harwell. We have developed a range of highly specialized technologies incorporating robotic systems to enable the high throughput expression, purification and crystallization of recombinant proteins. In partnership with UK academic groups, this technology platform is being used to study a wide variety of proteins of biomedical interest. To see the OPPF-UK in action please follow the link: http://www.youtube.com/watch?v=joWNZDBqrlQ.

The OPPF-UK is open to all UK academic groups and the use of our facilities is "free at the point of access" following approval of projects by independent reviewers. Access is largely delivered through working visits to the OPPF-UK by PhD students and PRDAs providing hands-on training and the opportunity of transferring methods to their own laboratories.

Technology development in the OPPF-UK is focused on streamlining the production of recombinant proteins, particularly membrane proteins and protein complexes. Research interests include structural enzymology and the use of protein-based ligands to disrupt protein: protein interactions.

Name Department Institution Country
Dr Floriano Paes FIOCRUZ-IOC Brazil
Dr Nick Furnham London School of Hygiene and Tropical Medicine United Kingdom
Dr Darren Tomlinson Bioscreening Technology Group, School of Molecular and Cellular Biology University of Leeds United Kingdom
Awanye AM, Chang C-M, Wheeler JX, Chan H, Marsay L, Dold C, Rollier CS, Bird LE, Nettleship JE, Owens RJ et al. 2019. Immunogenicity profiling of protein antigens from capsular group B Neisseria meningitidis. Sci Rep, 9 (1), pp. 6843. | Show Abstract | Read more

Outer membrane vesicle (OMV)- based vaccines have been used to provide strain-specific protection against capsular group B Neisseria meningitidis infections, but the full breadth of the immune response against the components of the OMV has not been established. Sera from adults vaccinated with an OMV vaccine were used to screen 91 outer membrane proteins (OMPs) incorporated in an antigen microarray panel. Antigen-specific IgG levels were quantified pre-vaccination, and after 12 and 18 weeks. These results were compared with IgG levels from mice vaccinated with the same OMV vaccine. The repertoires of highly responding antigens in humans and mice overlapped, but were not identical. The highest responding antigens to human IgG comprised four integral OMPs (PorA, PorB, OpcA and PilQ), a protein which promotes the stability of PorA and PorB (RmpM) and two lipoproteins (BamC and GNA1162). These observations will assist in evaluating the role of minor antigen components within OMVs in providing protection against meningococcal infection. In addition, the relative dominance of responses to integral OMPs in humans emphasizes the importance of this subclass and points to the value of maintaining conformational epitopes from integral membrane proteins in vaccine formulations.

Ahmed AR, Owens RJ, Stubbs CD, Parker AW, Hitchman R, Yadav RB, Dumoux M, Hawes C, Botchway SW. 2019. Direct imaging of the recruitment and phosphorylation of S6K1 in the mTORC1 pathway in living cells. Sci Rep, 9 (1), pp. 3408. | Show Abstract | Read more

Knowledge of protein signalling pathways in the working cell is seen as a primary route to identifying and developing targeted medicines. In recent years there has been a growing awareness of the importance of the mTOR pathway, making it an attractive target for therapeutic intervention in several diseases. Within this pathway we have focused on S6 kinase 1 (S6K1), the downstream phosphorylation substrate of mTORC1, and specifically identify its juxtaposition with mTORC1. When S6K1 is co-expressed with raptor we show that S6K1 is translocated from the nucleus to the cytoplasm. By developing a novel biosensor we demonstrate in real-time, that phosphorylation and de-phosphorylation of S6K1 occurs mainly in the cytoplasm of living cells. Furthermore, we show that the scaffold protein raptor, that typically recruits mTOR substrates, is not always involved in S6K1 phosphorylation. Overall, we demonstrate how FRET-FLIM imaging technology can be used to show localisation of S6K1 phosphorylation in living cells and hence a key site of action of inhibitors targeting mTOR phosphorylation.

Romanello L, Zeraik AE, de Freitas Fernandes A, Torini JR, Bird LE, Nettleship JE, Rada H, Reddivari Y, Owens RJ, Serrão VHB et al. 2019. In vitro and in vivo characterization of the multiple isoforms of Schistosoma mansoni hypoxanthine-guanine phosphoribosyltransferases. Mol Biochem Parasitol, 229 pp. 24-34. | Show Abstract | Read more

Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the "de novo" purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8 Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.

Sabaratnam K, Renner M, Paesen G, Harlos K, Nair V, Owens RJ, Grimes JM. 2019. Insights from the crystal structure of the chicken CREB3 bZIP suggest that members of the CREB3 subfamily transcription factors may be activated in response to oxidative stress. Protein Sci, 28 (4), pp. 779-787. | Show Abstract | Read more

cAMP response element binding Protein 3 (CREB3) is an endoplasmic reticulum (ER) membrane-bound transcription factor, which belongs to the basic leucine zipper (bZIP) superfamily of eukaryotic transcription factors. CREB3 plays a role in the ER-stress induced unfolded protein response (UPR) and is a multifunctional cellular factor implicated in a number of biological processes including cell proliferation and migration, tumor suppression, and immune-related gene expression. To gain structural insights into the transcription factor, we determined the crystal structure of the conserved bZIP domain of chicken CREB3 (chCREB3) to a resolution of 3.95 Å. The X-ray structure provides evidence that chCREB3 can form a stable homodimer. The chCREB3 bZIP has a structured, pre-formed DNA binding region, even in the absence of DNA, a feature that could potentially enhance both the DNA binding specificity and affinity of chCREB3. Significantly, the homodimeric bZIP possesses an intermolecular disulfide bond that connects equivalent cysteine residues of the parallel helices in the leucine zipper region. This disulfide bond in the hydrophobic core of the bZIP may increase the stability of the homodimer under oxidizing conditions. Moreover, sequence alignment of bZIP sequences from chicken, human, and mouse reveals that only members of the CREB3 subfamily contain this cysteine residue, indicating that it could act as a redox-sensor. Taken together, these results suggest that the activity of these transcription factors may be redox-regulated and they may be activated in response to oxidative stress.

Kim YC, Lopez-Camacho C, Nettleship JE, Rahman N, Hill ML, Silva-Reyes L, Ortiz-Martinez G, Figueroa-Aguilar G, Mar MA, Vivanco-Cid H et al. 2018. Optimization of Zika virus envelope protein production for ELISA and correlation of antibody titers with virus neutralization in Mexican patients from an arbovirus endemic region. Virol J, 15 (1), pp. 193. | Show Abstract | Read more

BACKGROUND: Zika virus (ZIKV) has become a global threat with immediate need for accurate diagnostics, efficacious vaccines and therapeutics. Several ZIKV envelope (Env)-based vaccines have been developed recently. However, many commercially available ZIKV Env are based on the African lineage and produced in insect cells. Here, we sought to produce Asian-lineage ZIKV Env in mammalian cells for research and clinical applications. METHODS: We designed various gene expression constructs to optimize the production of ZIKV using prM-Env and full or C-terminal truncations of Env; with or without a rat CD4 fusion partner to allow large-scale production of soluble protein in mammalian HEK293 cells. Protein expression was verified by mass spectrometry and western-blot with a pan-flavivirus antibody, a ZIKV Env monoclonal antibody and with immune sera from adenoviral (ChAdOx1) ZIKV Env-vaccinated mice. The resulting Env-CD4 was used as a coating reagent for immunoassay (ELISA) using both mouse and human seropositive sera. RESULTS: Replacement of the C-terminus transmembrane Env domain by a rat CD4 and addition of prM supported optimal expression and secretion of Env. Binding between the antigens and the antibodies was similar to binding when using commercially available ZIKV Env reagents. Furthermore, antibodies from ZIKV patients bound ZIKV Env-CD4 in ELISA assays, whereas sera from healthy blood donors yielded minimal OD background. The serological outcomes of this assay correlated also with ZIKV neutralisation capacity in vitro. CONCLUSIONS: Results obtained from this study indicate the potential of the Asian-lineage Zika Env-CD4 and Env proteins in ELISA assays to monitor humoral immune responses in upcoming clinical trials as well as a sero-diagnostic tool in ZIKV infection.

Salimraj R, Hinchliffe P, Kosmopoulou M, Tyrrell JM, Brem J, van Berkel SS, Verma A, Owens RJ, McDonough MA, Walsh TR et al. 2019. Crystal structures of VIM-1 complexes explain active site heterogeneity in VIM-class metallo-β-lactamases. FEBS J, 286 (1), pp. 169-183. | Show Abstract | Read more

Metallo-β-Lactamases (MBLs) protect bacteria from almost all β-lactam antibiotics. Verona integron-encoded MBL (VIM) enzymes are among the most clinically important MBLs, with VIM-1 increasing in carbapenem-resistant Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae) that are among the hardest bacterial pathogens to treat. VIM enzymes display sequence variation at residues (224 and 228) that in related MBLs are conserved and participate in substrate binding. How they accommodate this variability, while retaining catalytic efficiency against a broad substrate range, has remained unclear. Here, we present crystal structures of VIM-1 and its complexes with a substrate-mimicking thioenolate inhibitor, ML302F, that restores meropenem activity against a range of VIM-1 producing clinical strains, and the hydrolysed product of the carbapenem meropenem. Comparison of these two structures identifies a water-mediated hydrogen bond, between the carboxylate group of substrate/inhibitor and the backbone carbonyl of the active site zinc ligand Cys221, that is common to both complexes. Structural comparisons show that the responsible Cys221-bound water is observed in all known VIM structures, participates in carboxylate binding with other inhibitor classes, and thus effectively replicates the role of the conserved Lys224 in analogous complexes with other MBLs. These results provide a mechanism for substrate binding that permits the variation at positions 224 and 228 that is a hallmark of VIM MBLs. ENZYMES: EC 3.5.2.6 DATABASES: Co-ordinates and structure factors for protein structures described in this manuscript have been deposited in the Protein Data Bank (www.rcsb.org/pdb) with accession codes 5N5G (VIM-1), 5N5H (VIM-1:ML302F complex) and 5N5I (VIM-1-hydrolysed meropenem complex).

Torini JR, Romanello L, Batista FAH, Serrão VHB, Faheem M, Zeraik AE, Bird L, Nettleship J, Reddivari Y, Owens R et al. 2018. The molecular structure of Schistosoma mansoni PNP isoform 2 provides insights into the nucleoside selectivity of PNPs. PLoS One, 13 (9), pp. e0203532. | Show Abstract | Read more

Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 μM for cytosine, and a KM of 76.3 μM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.

Shimmon G, Kotecha A, Ren J, Asfor AS, Newman J, Berryman S, Cottam EM, Gold S, Tuthill TJ, King DP et al. 2018. Generation and characterisation of recombinant FMDV antibodies: Applications for advancing diagnostic and laboratory assays. PLoS One, 13 (8), pp. e0201853. | Show Abstract | Read more

Foot-and-mouth disease (FMD) affects economically important livestock and is one of the most contagious viral diseases. The most commonly used FMD diagnostic assay is a sandwich ELISA. However, the main disadvantage of this ELISA is that it requires anti-FMD virus (FMDV) serotype-specific antibodies raised in small animals. This problem can be, in part, overcome by using anti-FMDV monoclonal antibodies (MAbs) as detecting reagents. However, the long-term use of MAbs may be problematic and they may need to be replaced. Here we have constructed chimeric antibodies (mouse/rabbit D9) and Fabs (fragment antigen-binding) (mouse/cattle D9) using the Fv (fragment variable) regions of a mouse MAb, D9 (MAb D9), which recognises type O FMDV. The mouse/rabbit D9 chimeric antibody retained the FMDV serotype-specificity of MAb D9 and performed well in a FMDV detection ELISA as well as in routine laboratory assays. Cryo-electron microscopy analysis confirmed engagement with antigenic site 1 and peptide competition studies identified the aspartic acid at residue VP1 147 as a novel component of the D9 epitope. This chimeric expression approach is a simple but effective way to preserve valuable FMDV antibodies, and has the potential for unlimited generation of antibodies and antibody fragments in recombinant systems with the concomitant positive impacts on the 3Rs (Replacement, Reduction and Refinement) principles.

Trundová M, Kovaľ T, Owens RJ, Fejfarová K, Dušková J, Kolenko P, Dohnálek J. 2018. Highly stable single-strand-specific 3'-nuclease/nucleotidase from Legionella pneumophila. Int J Biol Macromol, 114 pp. 776-787. | Show Abstract | Read more

The Gram-negative bacterium Legionella pneumophila is one of the known opportunistic human pathogens with a gene coding for a zinc-dependent S1-P1 type nuclease. Bacterial zinc-dependent 3'-nucleases/nucleotidases are little characterized and not fully understood, including L. pneumophila nuclease 1 (Lpn1), in contrast to many eukaryotic representatives with in-depth studies available. To help explain the principle properties and role of these enzymes in intracellular prokaryotic pathogens we have designed and optimized a heterologous expression protocol utilizing E. coli together with an efficient purification procedure, and performed detailed characterization of the enzyme. Replacement of Ni2+ ions by Zn2+ ions in affinity purification proved to be a crucial step in the production of pure and stable protein. The production protocol provides protein with high yield, purity, stability, and solubility for structure-function studies. We show that highly thermostable Lpn1 is active mainly towards RNA and ssDNA, with pH optima 7.0 and 6.0, respectively, with low activity towards dsDNA; the enzyme features pronounced substrate inhibition. Bioinformatic and experimental analysis, together with computer modeling and electrostatics calculations point to an unusually high positive charge on the enzyme surface under optimal conditions for catalysis. The results help explain the catalytic properties of Lpn1 and its substrate inhibition.

Trundova M, Koval T, Owens RJ, Fejfarova K, Duskova J, Kolenko P, Dohnalek J. 2018. Highly stable single-strand-specific 3'-nuclease/nucleotidase from Legionella pneumophila INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES, 114 pp. 776-787. | Read more

Hardman CS, Chen Y-L, Salimi M, Jarrett R, Johnson D, Järvinen VJ, Owens RJ, Repapi E, Cousins DJ, Barlow JL et al. 2017. CD1a presentation of endogenous antigens by group 2 innate lymphoid cells. Sci Immunol, 2 (18), pp. eaan5918-eaan5918. | Show Abstract | Read more

Group 2 innate lymphoid cells (ILC2) are effectors of barrier immunity, with roles in infection, wound healing, and allergy. A proportion of ILC2 express MHCII (major histocompatibility complex II) and are capable of presenting peptide antigens to T cells and amplifying the subsequent adaptive immune response. Recent studies have highlighted the importance of CD1a-reactive T cells in allergy and infection, activated by the presentation of endogenous neolipid antigens and bacterial components. Using a human skin challenge model, we unexpectedly show that human skin-derived ILC2 can express CD1a and are capable of presenting endogenous antigens to T cells. CD1a expression is up-regulated by TSLP (thymic stromal lymphopoietin) at levels observed in the skin of patients with atopic dermatitis, and the response is dependent on PLA2G4A. Furthermore, this pathway is used to sense Staphylococcus aureus by promoting Toll-like receptor-dependent CD1a-reactive T cell responses to endogenous ligands. These findings define a previously unrecognized role for ILC2 in lipid surveillance and identify shared pathways of CD1a- and PLA2G4A-dependent ILC2 inflammation amenable to therapeutic intervention.

Robinson JI, Baxter EW, Owen RL, Thomsen M, Tomlinson DC, Waterhouse MP, Win SJ, Nettleship JE, Tiede C, Foster RJ et al. 2018. Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action. Proc Natl Acad Sci U S A, 115 (1), pp. E72-E81. | Show Abstract | Read more

Protein-protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called "Affimer" that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.

Tiede C, Bedford R, Heseltine SJ, Smith G, Wijetunga I, Ross R, AlQallaf D, Roberts AP, Balls A, Curd A et al. 2017. Affimer proteins are versatile and renewable affinity reagents. Elife, 6 | Show Abstract | Read more

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

West NR, Hegazy AN, Owens BMJ, Bullers SJ, Linggi B, Buonocore S, Coccia M, Görtz D, This S, Stockenhuber K et al. 2017. Oncostatin M drives intestinal inflammation and predicts response to tumor necrosis factor-neutralizing therapy in patients with inflammatory bowel disease. Nat Med, 23 (5), pp. 579-589. | Show Abstract | Read more

Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are complex chronic inflammatory conditions of the gastrointestinal tract that are driven by perturbed cytokine pathways. Anti-tumor necrosis factor-α (TNF) antibodies are mainstay therapies for IBD. However, up to 40% of patients are nonresponsive to anti-TNF agents, which makes the identification of alternative therapeutic targets a priority. Here we show that, relative to healthy controls, inflamed intestinal tissues from patients with IBD express high amounts of the cytokine oncostatin M (OSM) and its receptor (OSMR), which correlate closely with histopathological disease severity. The OSMR is expressed in nonhematopoietic, nonepithelial intestinal stromal cells, which respond to OSM by producing various proinflammatory molecules, including interleukin (IL)-6, the leukocyte adhesion factor ICAM1, and chemokines that attract neutrophils, monocytes, and T cells. In an animal model of anti-TNF-resistant intestinal inflammation, genetic deletion or pharmacological blockade of OSM significantly attenuates colitis. Furthermore, according to an analysis of more than 200 patients with IBD, including two cohorts from phase 3 clinical trials of infliximab and golimumab, high pretreatment expression of OSM is strongly associated with failure of anti-TNF therapy. OSM is thus a potential biomarker and therapeutic target for IBD, and has particular relevance for anti-TNF-resistant patients.

Romanello L, Serrão VHB, Torini JR, Bird LE, Nettleship JE, Rada H, Reddivari Y, Owens RJ, DeMarco R, Brandão-Neto J, Pereira HD. 2017. Structural and kinetic analysis of Schistosoma mansoni Adenylosuccinate Lyase (SmADSL). Mol Biochem Parasitol, 214 pp. 27-35. | Show Abstract | Read more

Schistosoma mansoni is the parasite responsible for schistosomiasis, a disease that affects about 218 million people worldwide. Currently, both direct treatment and disease control initiatives rely on chemotherapy using a single drug, praziquantel. Concerns over the possibility of resistance developing to praziquantel, have stimulated efforts to develop new drugs for the treatment of schistosomiasis. Schistosomes do not have the de novo purine biosynthetic pathway, and instead depend entirely on the purine salvage pathway to supply its need for purines. The purine salvage pathway has been reported as a potential target for developing new drugs against schistosomiasis. Adenylosuccinate lyase (SmADSL) is an enzyme in this pathway, which cleaves adenylosuccinate (ADS) into adenosine 5'-monophosphate (AMP) and fumarate. SmADSL kinetic characterization was performed by isothermal titration calorimetry (ITC) using both ADS and SAICAR as substrates. Structures of SmADSL in Apo form and in complex with AMP were elucidated by x-ray crystallography revealing a highly conserved tetrameric structure required for their function since the active sites are formed from residues of three different subunits. The active sites are also highly conserved between species and it is difficult to identify a potent species-specific inhibitor for the development of new therapeutic agents. In contrast, several mutagenesis studies have demonstrated the importance of dimeric interface residues in the stability of the quaternary structure of the enzyme. The lower conservation of these residues between SmADSL and human ADSL could be used to lead the development of anti-schistosomiasis drugs based on disruption of subunit interfaces. These structures and kinetics data add another layer of information to Schistosoma mansoni purine salvage pathway.

Roatsch M, Robaa D, Pippel M, Nettleship JE, Reddivari Y, Bird LE, Hoffmann I, Franz H, Owens RJ, Schüle R et al. 2016. Substituted 2-(2-aminopyrimidin-4-yl)pyridine-4-carboxylates as potent inhibitors of JumonjiC domain-containing histone demethylases. Future Med Chem, 8 (13), pp. 1553-1571. | Show Abstract | Read more

BACKGROUND: Aberrant expression of iron(II)- and 2-oxoglutarate-dependent JumonjiC histone demethylases has been linked to cancer. Potent demethylase inhibitors are drug candidates and biochemical tools to elucidate the functional impact of demethylase inhibition. METHODS & RESULTS: Virtual screening identified a novel lead scaffold against JMJD2A with low-micromolar potency in vitro. Analogs were acquired from commercial sources respectively synthesized in feedback with biological testing. Optimized compounds were transformed into cell-permeable prodrugs. A cocrystal x-ray structure revealed the mode of binding of these compounds as competitive to 2-oxoglutarate and confirmed kinetic experiments. Selectivity studies revealed a preference for JMJD2A and JARID1A over JMJD3. CONCLUSION: Virtual screening and rational structural optimization led to a novel scaffold for highly potent and selective JMJD2A inhibitors.

Lanyon-Hogg T, Masumoto N, Bodakh G, Konitsiotis AD, Thinon E, Rodgers UR, Owens RJ, Magee AI, Tate EW. 2016. Synthesis and characterisation of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine inhibitors of Hedgehog acyltransferase. Data Brief, 7 pp. 257-281. | Show Abstract | Read more

In this data article we describe synthetic and characterisation data for four members of the 5-acyl-6,7-dihydrothieno[3,2-c]pyridine (termed "RU-SKI") class of inhibitors of Hedgehog acyltransferase, including associated NMR spectra for final compounds. RU-SKI compounds were selected for synthesis based on their published high potencies against the enzyme target. RU-SKI 41 (9a), RU-SKI 43 (9b), RU-SKI 101 (9c), and RU-SKI 201 (9d) were profiled for activity in the related article "Click chemistry armed enzyme linked immunosorbent assay to measure palmitoylation by Hedgehog acyltransferase" (Lanyon-Hogg et al., 2015) [1]. (1)H NMR spectral data indicate different amide conformational ratios between the RU-SKI inhibitors, as has been observed in other 5-acyl-6,7-dihydrothieno[3,2-c]pyridines. The synthetic and characterisation data supplied in the current article provide validated access to the class of RU-SKI inhibitors.

Owens R. 2016. Methods in integrated structural biology. Methods, 95 pp. 1-2. | Read more

Ren J, Nettleship JE, Males A, Stuart DI, Owens RJ. 2016. Crystal structures of penicillin-binding protein 3 in complexes with azlocillin and cefoperazone in both acylated and deacylated forms. FEBS Lett, 590 (2), pp. 288-297. | Show Abstract | Read more

Penicillin-binding protein 3 (PBP3) from Pseudomonas aeruginosa is the molecular target of β-lactam-based antibiotics. Structures of PBP3 in complexes with azlocillin and cefoperazone, which are in clinical use for the treatment of pseudomonad infections, have been determined to 2.0 Å resolution. Together with data from other complexes, these structures identify a common set of residues involved in the binding of β-lactams to PBP3. Comparison of wild-type and an active site mutant (S294A) showed that increased thermal stability of PBP3 following azlocillin binding was entirely due to covalent binding to S294, whereas cefoperazone binding produces some increase in stability without the covalent link. Consistent with this, a third crystal structure was determined in which the hydrolysis product of cefoperazone was noncovalently bound in the active site of PBP3. This is the first structure of a complex between a penicillin-binding protein and cephalosporic acid and may be important in the design of new noncovalent PBP3 inhibitors.

Bird LE, Nettleship JE, Järvinen V, Rada H, Verma A, Owens RJ. 2016. Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters. Adv Exp Med Biol, 922 pp. 1-11. | Show Abstract | Read more

The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years.

Lanyon-Hogg T, Masumoto N, Bodakh G, Konitsiotis AD, Thinon E, Rodgers UR, Owens RJ, Magee AI, Tate EW. 2015. Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase. Anal Biochem, 490 pp. 66-72. | Show Abstract | Read more

Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl β-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.

Arena de Souza V, Scott DJ, Nettleship JE, Rahman N, Charlton MH, Walsh MA, Owens RJ. 2015. Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1. PLoS One, 10 (12), pp. e0143919. | Show Abstract | Read more

Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.

Lukacik P, Lobley CMC, Bumann M, Arena de Souza V, Owens RJ, O'Toole PW, Walsh MA. 2015. High-resolution structures of Lactobacillus salivarius transketolase in the presence and absence of thiamine pyrophosphate. Acta Crystallogr F Struct Biol Commun, 71 (Pt 10), pp. 1327-1334. | Show Abstract | Read more

Probiotic bacterial strains have been shown to enhance the health of the host through a range of mechanisms including colonization, resistance against pathogens, secretion of antimicrobial compounds and modulation of the activity of the innate immune system. Lactobacillus salivarius UCC118 is a well characterized probiotic strain which survives intestinal transit and has many desirable host-interaction properties. Probiotic bacteria display a wide range of catabolic activities, which determine their competitiveness in vivo. Some lactobacilli are heterofermentative and can metabolize pentoses, using a pathway in which transketolase and transaldolase are key enzymes. L. salivarius UCC118 is capable of pentose utilization because it encodes the key enzymes on a megaplasmid. The crystal structures of the megaplasmid-encoded transketolase with and without the enzyme cofactor thiamine pyrophosphate have been determined. Comparisons with other known transketolase structures reveal a high degree of structural conservation in both the catalytic site and the overall conformation. This work extends structural knowledge of the transketolases to the industrially and commercially important Lactobacillus genus.

Liu C, Zhao Y, He W, Wang W, Chen Y, Zhang S, Ma Y, Gohda J, Ishida T, Walter TS et al. 2015. A RANKL mutant used as an inter-species vaccine for efficient immunotherapy of osteoporosis. Sci Rep, 5 (1), pp. 14150. | Show Abstract | Read more

Anti-cytokine therapeutic antibodies have been demonstrated to be effective in the treatment of several auto-immune disorders. However, The problems in antibody manufacture and the immunogenicity caused by multiple doses of antibodies inspire people to use auto-cytokine as immunogen to induce anti-cytokine antibodies. Nevertheless, the tolerance for inducing immune response against self-antigen has hindered the wide application of the strategy. To overcome the tolerance, here we proposed a strategy using the inter-species cytokine as immunogen for active immunization (TISCAI) to induce anti-cytokine antibody. As a proof of concept, an inter-species cytokine RANKL was successfully used as immunogen to induce anti-RANKL immune response. Furthermore, to prevent undesirable side-effects, the human RANKL was mutated based on the crystal structure of the complex of human RANKL and its rodent counterpart receptor RANK. We found, the antibodies produced blocked the osteoclast development in vitro and osteoporosis in OVX rat models. The results demonstrated this strategy adopted is very useful for general anti-cytokine immunotherapy for different diseases settings.

Beale JH, Parker JL, Samsudin F, Barrett AL, Senan A, Bird LE, Scott D, Owens RJ, Sansom MSP, Tucker SJ et al. 2015. Crystal Structures of the Extracellular Domain from PepT1 and PepT2 Provide Novel Insights into Mammalian Peptide Transport. Structure, 23 (10), pp. 1889-1899. | Show Abstract | Read more

Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.

Beale JH, Parker JL, Samsudin F, Barrett AL, Senan A, Bird LE, Scott D, Owens RJ, Sansom MSP, Tucker SJ et al. 2015. Crystal structures of the extracellular domain from PepT1 and PepT2 provide novel insights into mammalian peptide transport Structure, 23 (10), pp. 1889-1899. | Show Abstract | Read more

© 2015 The Authors. Mammals obtain nitrogen via the uptake of di- and tri-peptides in the gastrointestinal tract through the action of PepT1 and PepT2, which are members of the POT family of proton-coupled oligopeptide transporters. PepT1 and PepT2 also play an important role in drug transport in the human body. Recent crystal structures of bacterial homologs revealed a conserved peptide-binding site and mechanism of transport. However, a key structural difference exists between bacterial and mammalian homologs with only the latter containing a large extracellular domain, the function of which is currently unknown. Here, we present the crystal structure of the extracellular domain from both PepT1 and PepT2 that reveal two immunoglobulin-like folds connected in tandem, providing structural insight into mammalian peptide transport. Functional and biophysical studies demonstrate that these domains interact with the intestinal protease trypsin, suggesting a role in clustering proteolytic activity to the site of peptide transport in eukaryotic cells.

Bird LE, Rada H, Verma A, Gasper R, Birch J, Jennions M, Lӧwe J, Moraes I, Owens RJ. 2015. Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli. J Vis Exp, (95), pp. e52357. | Show Abstract | Read more

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Owens RJ. 2015. Structural proteomics: high-throughput methods. Preface Methods in molecular biology (Clifton, N.J.), 1261 pp. v.

Hu N-J, Rada H, Rahman N, Nettleship JE, Bird L, Iwata S, Drew D, Cameron AD, Owens RJ. 2015. GFP-based expression screening of membrane proteins in insect cells using the baculovirus system. Methods Mol Biol, 1261 pp. 197-209. | Show Abstract | Read more

A key step in the production of recombinant membrane proteins for structural studies is the optimization of protein yield and quality. One commonly used approach is to fuse the protein to green fluorescent protein (GFP), enabling expression to be tracked without the need to purify the protein. Combining fusion to green fluorescent protein with the baculovirus expression system provides a useful platform for both screening and production of eukaryotic membrane proteins. In this chapter we describe our protocol for the expression screening of membrane proteins in insect cells using fusion to GFP as a reporter. We use both SDS-PAGE with in-gel fluorescence imaging and fluorescence-detection size-exclusion chromatography (FSEC) to screen for expression.

Owens RJ. 2015. Structural proteomics: high-throughput methods. Preface. Methods Mol Biol, 1261 pp. v. | Show Abstract | Read more

© Springer Science+Business Media New York 2015. All rights are reserved. This updated and expanded volume reflects the current state of the structural protein field with improved and refined protocols that have been applied to particularly challenging proteins, notably integral membrane proteins and multi-protein complexes. Structural Proteomics: High-Throughput Methods, Second Edition begins by exploring the resources available for curation, annotation, and structure prediction in silico, and continues with methods for sample preparation of both proteins and crystals, as well as structural characterization techniques. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Practical and up-to-date, Structural Proteomics: High-Throughput Methods, Second Edition will aid researchers in expanding our knowledge of this vital and expansive area of protein science.

Nettleship JE, Watson PJ, Rahman-Huq N, Fairall L, Posner MG, Upadhyay A, Reddivari Y, Chamberlain JMG, Kolstoe SE, Bagby S et al. 2015. Transient expression in HEK 293 cells: an alternative to E. coli for the production of secreted and intracellular mammalian proteins. Methods Mol Biol, 1258 pp. 209-222. | Show Abstract | Read more

Transient transfection of human embryonic kidney cells (HEK 293) enables the rapid and affordable lab-scale production of recombinant proteins. In this chapter protocols for the expression and purification of both secreted and intracellular proteins using transient expression in HEK 293 cells are described.

Tiede C, Tang AAS, Deacon SE, Mandal U, Nettleship JE, Owen RL, George SE, Harrison DJ, Owens RJ, Tomlinson DC, McPherson MJ. 2014. Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications. Protein Eng Des Sel, 27 (5), pp. 145-155. | Show Abstract | Read more

We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 10(10) clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications.

Bird LE, Rada H, Flanagan J, Diprose JM, Gilbert RJC, Owens RJ. 2014. Application of In-Fusion™ cloning for the parallel construction of E. coli expression vectors. Methods Mol Biol, 1116 pp. 209-234. | Show Abstract | Read more

In-Fusion™ cloning is a flexible DNA ligase-independent cloning technology that has wide-ranging uses in molecular biology. In this chapter we describe the protocols used in the OPPF-UK to design and construct expression vectors using In-Fusion™. Our method for small scale expression screening in Escherichia coli of constructs generated by In-Fusion™ is also outlined.

Bird LE, Rada H, Flanagan J, Diprose JM, Gilbert RJC, Owens RJ. 2014. Application of In-Fusion™ cloning for the parallel construction of E. coli expression vectors Methods in Molecular Biology, 1116 pp. 209-234. | Show Abstract | Read more

In-Fusion™ cloning is a fl exible DNA ligase-independent cloning technology that has wide-ranging uses in molecular biology. In this chapter we describe the protocols used in the OPPF-UK to design and construct expression vectors using In-Fusion™. Our method for small scale expression screening in Escherichia coli of constructs generated by In-Fusion™ is also outlined. © Springer Science+Business Media New York 2014.

Makena A, van Berkel SS, Lejeune C, Owens RJ, Verma A, Salimraj R, Spencer J, Brem J, Schofield CJ. 2013. Chromophore-linked substrate (CLS405): probing metallo-β-lactamase activity and inhibition. ChemMedChem, 8 (12), pp. 1923-1929. | Show Abstract | Read more

Serine- and metallo-β-lactamases present a threat to the clinical use of nearly all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. Efforts to develop metallo-β-lactamase (MBL) inhibitors require suitable screening platforms to allow the rapid determination of β-lactamase activity and efficient inhibition. Unfortunately, the platforms currently available are not ideal for this purpose. Further progress in MBL inhibitor identification requires inexpensive and widely applicable assays. Herein the identification of an inexpensive and stable chromogenic substrate suitable for use in assays of clinically relevant MBLs is described. (6R,7R)-3-((4-Nitrophenoxy)methyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 5,5-dioxide (CLS405) was synthesised in a three-step protocol. CLS405 was then characterised spectroscopically, and its stability and kinetic properties evaluated. With a Δλmax value of 100 nm between the parent and hydrolysis product, a higher analytical accuracy is possible with CLS405 than with commonly used chromogenic substrates. The use of CLS405 in assays was validated by MBL activity measurements and inhibitor screening that resulted in the identification of N-hydroxythiazoles as new inhibitor scaffolds for MBLs. Further evaluation of the identified N-hydroxythiazoles against a panel of clinically relevant MBLs showed that they possess inhibitory activities in the mid- to low-micromolar range. The findings of this study provide both a useful tool compound for further inhibitor identification, and novel scaffolds for the design of improved MBL inhibitors with potential as antibiotics against resistant strains of bacteria.

Knox R, Nettleship JE, Chang VT, Hui ZK, Santos AM, Rahman N, Ho L-P, Owens RJ, Davis SJ. 2013. A streamlined implementation of the glutamine synthetase-based protein expression system. BMC Biotechnol, 13 (1), pp. 74. | Show Abstract | Read more

BACKGROUND: The glutamine synthetase-based protein expression system is widely used in industry and academia for producing recombinant proteins but relies on the cloning of transfected cells, necessitating substantial investments in time and handling. We streamlined the production of protein-producing cultures of Chinese hamster ovary cells using this system by co-expressing green fluorescent protein from an internal ribosomal entry site and selecting for high green fluorescent protein-expressing cells using fluorescence-activated cell sorting. RESULTS: Whereas other expression systems utilizing green fluorescent protein and fluorescence-activated cell sorting-based selection have relied on two or more sorting steps, we obtained stable expression of a test protein at levels >50% of that of an "average" clone and ~40% that of the "best" clone following a single sorting step. Versus clone-based selection, the principal savings are in the number of handling steps (reduced by a third), handling time (reduced by 70%), and the time needed to produce protein-expressing cultures (reduced by ~3 weeks). Coupling the glutamine synthetase-based expression system with product-independent selection in this way also facilitated the production of a hard-to-assay protein. CONCLUSION: Utilizing just a single fluorescence-activated cell sorting-based selection step, the new streamlined implementation of the glutamine synthetase-based protein expression system offers protein yields sufficient for most research purposes, where <10 mg/L of protein expression is often required but relatively large numbers of constructs frequently need to be trialed.

van Berkel SS, Nettleship JE, Leung IKH, Brem J, Choi H, Stuart DI, Claridge TDW, McDonough MA, Owens RJ, Ren J, Schofield CJ. 2013. Binding of (5S)-penicilloic acid to penicillin binding protein 3. ACS Chem Biol, 8 (10), pp. 2112-2116. | Show Abstract | Read more

β-Lactam antibiotics react with penicillin binding proteins (PBPs) to form relatively stable acyl-enzyme complexes. We describe structures derived from the reaction of piperacillin with PBP3 (Pseudomonas aeruginosa) including not only the anticipated acyl-enzyme complex but also an unprecedented complex with (5S)-penicilloic acid, which was formed by C-5 epimerization of the nascent (5R)-penicilloic acid product. Formation of the complex was confirmed by solution studies, including NMR. Together, these results will be useful in the design of new PBP inhibitors and raise the possibility that noncovalent PBP inhibition by penicilloic acids may be of clinical relevance.

van Berkel SS, Brem J, Rydzik AM, Salimraj R, Cain R, Verma A, Owens RJ, Fishwick CWG, Spencer J, Schofield CJ. 2013. Assay platform for clinically relevant metallo-β-lactamases. J Med Chem, 56 (17), pp. 6945-6953. | Show Abstract | Read more

Metallo-β-lactamases (MBLs) are a growing threat to the use of almost all clinically used β-lactam antibiotics. The identification of broad-spectrum MBL inhibitors is hampered by the lack of a suitable screening platform, consisting of appropriate substrates and a set of clinically relevant MBLs. We report procedures for the preparation of a set of clinically relevant metallo-β-lactamases (i.e., NDM-1 (New Delhi MBL), IMP-1 (Imipenemase), SPM-1 (São Paulo MBL), and VIM-2 (Verona integron-encoded MBL)) and the identification of suitable fluorogenic substrates (umbelliferone-derived cephalosporins). The fluorogenic substrates were compared to chromogenic substrates (CENTA, nitrocefin, and imipenem), showing improved sensitivity and kinetic parameters. The efficiency of the fluorogenic substrates was exemplified by inhibitor screening, identifying 4-chloroisoquinolinols as potential pan MBL inhibitors.

Nettleship JE, Ren J, Scott DJ, Rahman N, Hatherley D, Zhao Y, Stuart DI, Barclay AN, Owens RJ. 2013. Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment. BMC Struct Biol, 13 (1), pp. 13. | Show Abstract | Read more

BACKGROUND: Signal Regulatory Protein γ (SIRPγ) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRPγ is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRPγ. RESULTS: We obtained crystals of SIRPγ by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRPα and SIRPβ. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRPγ and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRPγ and SIRPα but not SIRPβ. Comparison of the three domain structures of SIRPγ and SIRPα showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRPγ in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRPγ were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRPγ: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd = 1.2 +/- 0.3 μM). CONCLUSION: The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRPγ stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution.

Byrne RT, Whelan F, Aller P, Bird LE, Dowle A, Lobley CMC, Reddivari Y, Nettleship JE, Owens RJ, Antson AA, Waterman DG. 2013. S-Adenosyl-S-carboxymethyl-L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA. Acta Crystallogr D Biol Crystallogr, 69 (Pt 6), pp. 1090-1098. | Show Abstract | Read more

Uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo(5)U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo(5)U and was annotated as an S-adenosyl-L-methionine-dependent (SAM-dependent) methyltransferase on the basis of its sequence homology to other SAM-containing enzymes. However, both the crystal structure of Escherichia coli CmoA at 1.73 Å resolution and mass spectrometry demonstrate that it contains a novel cofactor, S-adenosyl-S-carboxymethyl-L-homocysteine (SCM-SAH), in which the donor methyl group is substituted by a carboxymethyl group. The carboxyl moiety forms a salt-bridge interaction with Arg199 that is conserved in a large group of CmoA-related proteins but is not conserved in other SAM-containing enzymes. This raises the possibility that a number of enzymes that have previously been annotated as SAM-dependent are in fact SCM-SAH-dependent. Indeed, inspection of electron density for one such enzyme with known X-ray structure, PDB entry 1im8, suggests that the active site contains SCM-SAH and not SAM.

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Aricescu AR, Owens RJ. 2013. Expression of recombinant glycoproteins in mammalian cells: Towards an integrative approach to structural biology Current Opinion in Structural Biology, 23 (3), pp. 345-356. | Show Abstract | Read more

Mammalian cells are rapidly becoming the system of choice for the production of recombinant glycoproteins for structural biology applications. Their use has enabled the structural investigation of a whole new set of targets including large, multi-domain and highly glycosylated eukaryotic cell surface receptors and their supra-molecular assemblies. We summarize the technical advances that have been made in mammalian expression technology and highlight some of the structural insights that have been obtained using these methods. Looking forward, it is clear that mammalian cell expression will provide exciting and unique opportunities for an integrative approach to the structural study of proteins, especially of human origin and medically relevant, by bridging the gap between the purified state and the cellular context. © 2013 Elsevier Ltd.

Aricescu AR, Owens RJ. 2013. Expression of recombinant glycoproteins in mammalian cells: towards an integrative approach to structural biology. Curr Opin Struct Biol, 23 (3), pp. 345-356. | Show Abstract | Read more

Mammalian cells are rapidly becoming the system of choice for the production of recombinant glycoproteins for structural biology applications. Their use has enabled the structural investigation of a whole new set of targets including large, multi-domain and highly glycosylated eukaryotic cell surface receptors and their supra-molecular assemblies. We summarize the technical advances that have been made in mammalian expression technology and highlight some of the structural insights that have been obtained using these methods. Looking forward, it is clear that mammalian cell expression will provide exciting and unique opportunities for an integrative approach to the structural study of proteins, especially of human origin and medically relevant, by bridging the gap between the purified state and the cellular context.

Porta C, Xu X, Loureiro S, Paramasivam S, Ren J, Al-Khalil T, Burman A, Jackson T, Belsham GJ, Curry S et al. 2013. Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity. J Virol Methods, 187 (2), pp. 406-412. | Show Abstract | Read more

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

Cited:

33

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Porta C, Xu X, Loureiro S, Paramasivam S, Ren J, Al-Khalil T, Burman A, Jackson T, Belsham GJ, Curry S et al. 2013. Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity Journal of Virological Methods, 187 (2), pp. 406-412. | Show Abstract | Read more

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine. © 2012 Elsevier B.V.

Lobley CMC, Aller P, Douangamath A, Reddivari Y, Bumann M, Bird LE, Nettleship JE, Brandao-Neto J, Owens RJ, O'Toole PW, Walsh MA. 2012. Structure of ribose 5-phosphate isomerase from the probiotic bacteriumLactobacillus salivariusUCC118 Acta Crystallographica Section F Structural Biology and Crystallization Communications, 68 (12), pp. 1427-1433. | Show Abstract | Read more

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 A resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical and β d - ribose 5 - phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography. © 2012. © 2012 International Union of Crystallography All rights reserved.

Lobley CMC, Aller P, Douangamath A, Reddivari Y, Bumann M, Bird LE, Nettleship JE, Brandao-Neto J, Owens RJ, O'Toole PW, Walsh MA. 2012. Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118. Acta Crystallogr Sect F Struct Biol Cryst Commun, 68 (Pt 12), pp. 1427-1433. | Show Abstract | Read more

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

Yates LA, Lumb CN, Brahme NN, Zalyte R, Bird LE, De Colibus L, Owens RJ, Calderwood DA, Sansom MSP, Gilbert RJC. 2012. Structural and functional characterization of the kindlin-1 pleckstrin homology domain. J Biol Chem, 287 (52), pp. 43246-43261. | Show Abstract | Read more

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P(3); K(D) ∼100 μM) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P(3) as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P(2) on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P(3) is affected by the presence of PO(4) ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.

Sainsbury S, Ren J, Saunders NJ, Stuart DI, Owens RJ. 2012. Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) from Neisseria meningitidis. Acta Crystallogr Sect F Struct Biol Cryst Commun, 68 (Pt 7), pp. 730-737. | Show Abstract | Read more

The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from Neisseria meningitidis, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators.

Owen RL, Axford D, Nettleship JE, Owens RJ, Robinson JI, Morgan AW, Doré AS, Lebon G, Tate CG, Fry EE et al. 2012. Outrunning free radicals in room-temperature macromolecular crystallography. Acta Crystallogr D Biol Crystallogr, 68 (Pt 7), pp. 810-818. | Show Abstract | Read more

A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin γ Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A(2A) adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macromolecular crystallography.

Sainsbury S, Ren J, Saunders NJ, Stuart DI, Owens RJ. 2012. Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) fromNeisseria meningitidis Acta Crystallographica Section F Structural Biology and Crystallization Communications, 68 (7), pp. 730-737. | Show Abstract | Read more

<jats:p>The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from<jats:italic>Neisseria meningitidis</jats:italic>, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators.</jats:p>

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Owen RL, Axford D, Nettleship JE, Owens RJ, Robinson JI, Morgan AW, Doré AS, Lebon G, Tate CG, Fry EE et al. 2012. Outrunning free radicals in room-temperature macromolecular crystallography Acta Crystallographica Section D: Biological Crystallography, 68 (7), pp. 810-818. | Show Abstract | Read more

A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin γ Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A2A adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macro-molecular crystallography. © 2012 International Union of Crystallography. Printed in Singapore - all rights reserved.

Axford D, Owen RL, Aishima J, Foadi J, Morgan AW, Robinson JI, Nettleship JE, Owens RJ, Moraes I, Fry EE et al. 2012. In situ macromolecular crystallography using microbeams. Acta Crystallogr D Biol Crystallogr, 68 (Pt 5), pp. 592-600. | Show Abstract | Read more

Despite significant progress in high-throughput methods in macromolecular crystallography, the production of diffraction-quality crystals remains a major bottleneck. By recording diffraction in situ from crystals in their crystallization plates at room temperature, a number of problems associated with crystal handling and cryoprotection can be side-stepped. Using a dedicated goniometer installed on the microfocus macromolecular crystallography beamline I24 at Diamond Light Source, crystals have been studied in situ with an intense and flexible microfocus beam, allowing weakly diffracting samples to be assessed without a manual crystal-handling step but with good signal to noise, despite the background scatter from the plate. A number of case studies are reported: the structure solution of bovine enterovirus 2, crystallization screening of membrane proteins and complexes, and structure solution from crystallization hits produced via a high-throughput pipeline. These demonstrate the potential for in situ data collection and structure solution with microbeams.

Nettleship JE, Flanagan A, Rahman-Huq N, Hamer R, Owens RJ. 2012. Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells. Methods Mol Biol, 801 pp. 137-159. | Show Abstract | Read more

In this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5' primers that hybridise to the first framework sequence of the mouse light and heavy chains and 3' primers that bind to the heavy- and light-chain constant regions. The amplified sequences are inserted into mammalian cell expression vectors by In-Fusion™ cloning. This method allows vector and amplified DNA sequences to be seamlessly joined in a ligation-independent reaction. Transient co-expression of light-chain and heavy-chain genes in HEK 293T cells enables production of recombinant Fabs for functional and structural studies.

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WOS

Berry J-L, Phelan MM, Collins RF, Adomavicius T, Tonjum T, Frye SA, Bird L, Owens R, Ford RC, Lian L-Y, Derrick JP. 2012. Structure and Assembly of a Trans-Periplasmic Channel for Type IV Pili in Neisseria meningitidis PLOS PATHOGENS, 8 (9), | Read more

Wilke S, Groebe L, Maffenbeier V, Jäger V, Gossen M, Josewski J, Duda A, Polle L, Owens RJ, Wirth D et al. 2011. Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development PLoS ONE, 6 (12), | Show Abstract | Read more

Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP). © 2011 Wilke et al.

Wilke S, Groebe L, Maffenbeier V, Jäger V, Gossen M, Josewski J, Duda A, Polle L, Owens RJ, Wirth D et al. 2011. Streamlining homogeneous glycoprotein production for biophysical and structural applications by targeted cell line development. PLoS One, 6 (12), pp. e27829. | Show Abstract | Read more

Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP 'master' cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).

Green VL, Verma A, Owens RJ, Phillips SEV, Carr SB. 2011. Structure of New Delhi metallo-β-lactamase 1 (NDM-1). Acta Crystallogr Sect F Struct Biol Cryst Commun, 67 (Pt 10), pp. 1160-1164. | Show Abstract | Read more

Antibiotic resistance in bacterial pathogens poses a serious threat to human health and the metallo-β-lactamase (MBL) enzymes are responsible for much of this resistance. The recently identified New Delhi MBL 1 (NDM-1) is a novel member of this family that is capable of hydrolysing a wide variety of clinically important antibiotics. Here, the crystal structure of NDM-1 from Klebsiella pneumoniae is reported and its structure and active site are discussed in the context of other recently deposited coordinates of NDM-1.

Owens R. 2011. Methods in structural proteomics. Methods, 55 (1), pp. 1-2. | Read more

Daniel E, Lin B, Diprose JM, Griffiths SL, Morris C, Berry IM, Owens RJ, Blake R, Wilson KS, Stuart DI, Esnouf RM. 2011. xtalPiMS: a PiMS-based web application for the management and monitoring of crystallization trials. J Struct Biol, 175 (2), pp. 230-235. | Show Abstract | Read more

A major advance in protein structure determination has been the advent of nanolitre-scale crystallization and (in a high-throughput environment) the development of robotic systems for storing and imaging crystallization trials. Most of these trials are carried out in 96-well (or higher density) plates and managing them is a significant information management challenge. We describe xtalPiMS, a web-based application for the management and monitoring of crystallization trials. xtalPiMS has a user-interface layer based on the standards of the Protein Information Management System (PiMS) and a database layer which links the crystallization trial images to the meta-data associated with a particular crystallization trial. The user interface has been optimized for the efficient monitoring of high-throughput environments with three different automated imagers and work to support a fourth imager is in progress, but it can even be of use without robotics. The database can either be a PiMS database or a legacy database for which a suitable mapping layer has been developed.

Busso D, Peleg Y, Heidebrecht T, Romier C, Jacobovitch Y, Dantes A, Salim L, Troesch E, Schuetz A, Heinemann U et al. 2011. Expression of protein complexes using multiple Escherichia coli protein co-expression systems: a benchmarking study. J Struct Biol, 175 (2), pp. 159-170. | Show Abstract | Read more

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.

Holdom MD, Davies AM, Nettleship JE, Bagby SC, Dhaliwal B, Girardi E, Hunt J, Gould HJ, Beavil AJ, McDonnell JM et al. 2011. Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcɛRI. Nat Struct Mol Biol, 18 (5), pp. 571-576. | Show Abstract | Read more

Among antibody classes, IgE has a uniquely slow dissociation rate from, and high affinity for, its cell surface receptor FcɛRI. We show the structural basis for these key determinants of the ability of IgE to mediate allergic hypersensitivity through the 3.4-Å-resolution crystal structure of human IgE-Fc (consisting of the Cɛ2, Cɛ3 and Cɛ4 domains) bound to the extracellular domains of the FcɛRI α chain. Comparison with the structure of free IgE-Fc (reported here at a resolution of 1.9 Å) shows that the antibody, which has a compact, bent structure before receptor engagement, becomes even more acutely bent in the complex. Thermodynamic analysis indicates that the interaction is entropically driven, which explains how the noncontacting Cɛ2 domains, in place of the flexible hinge region of IgG antibodies, contribute together with the conformational changes to the unique binding properties of IgE.

Sainsbury S, Bird L, Rao V, Shepherd SM, Stuart DI, Hunter WN, Owens RJ, Ren J. 2011. Crystal structures of penicillin-binding protein 3 from Pseudomonas aeruginosa: comparison of native and antibiotic-bound forms. J Mol Biol, 405 (1), pp. 173-184. | Show Abstract | Read more

We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft. The orientations of the two β-lactams in the active site and the key interactions formed between the ligands and PBP3 are similar despite differences in the two drugs, indicating a degree of flexibility in the binding site. The conserved binding mode of β-lactam-based inhibitors appears to extend to other PBPs, as suggested by a comparison of the PBP3/ceftazidime complex and the Escherichia coli PBP1b/ceftoxamine complex. Since P. aeruginosa is an important human pathogen, the structural data reveal the mode of action of the frontline antibiotic ceftazidime at the molecular level. Improved drugs to combat infections by P. aeruginosa and related Gram-negative bacteria are sought and our study provides templates to assist that process and allows us to discuss new ways of inhibiting PBPs.

Bollati M, Alvarez K, Assenberg R, Baronti C, Canard B, Cook S, Coutard B, Decroly E, de Lamballerie X, Gould EA et al. 2010. Structure and functionality in flavivirus NS-proteins: perspectives for drug design. Antiviral Res, 87 (2), pp. 125-148. | Show Abstract | Read more

Flaviviridae are small enveloped viruses hosting a positive-sense single-stranded RNA genome. Besides yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as West Nile virus and dengue virus, are increasingly gaining attention due to their re-emergence and incidence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago flaviviruses were considered low interest candidates for drug design. At the start of the European Union VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex as a promising target for the development of antiviral compounds. Here we review structural and functional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the flavivirus replication complex. Most of the reviewed results were achieved within the European Union VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mechanisms. The ultimate aim of the reported approaches is to shed light on the design and development of antiviral drug leads.

Liu C, Walter TS, Huang P, Zhang S, Zhu X, Wu Y, Wedderburn LR, Tang P, Owens RJ, Stuart DI et al. 2010. Structural and functional insights of RANKL-RANK interaction and signaling. J Immunol, 184 (12), pp. 6910-6919. | Show Abstract | Read more

Bone remodeling involves bone resorption by osteoclasts and synthesis by osteoblasts and is tightly regulated by the receptor activator of the NF-kappaB ligand (RANKL)/receptor activator of the NF-kappaB (RANK)/osteoprotegerin molecular triad. RANKL, a member of the TNF superfamily, induces osteoclast differentiation, activation and survival upon interaction with its receptor RANK. The decoy receptor osteoprotegerin inhibits osteoclast formation by binding to RANKL. Imbalance in this molecular triad can result in diseases, including osteoporosis and rheumatoid arthritis. In this study, we report the crystal structures of unliganded RANK and its complex with RANKL and elucidation of critical residues for the function of the receptor pair. RANK represents the longest TNFR with four full cysteine-rich domains (CRDs) in which the CRD4 is stabilized by a sodium ion and a rigid linkage with CRD3. On association, RANK moves via a hinge region between the CRD2 and CRD3 to make close contact with RANKL; a significant structural change previously unseen in the engagement of TNFR superfamily 1A with its ligand. The high-affinity interaction between RANK and RANKL, maintained by continuous contact between the pair rather than the patched interaction commonly observed, is necessary for the function because a slightly reduced affinity induced by mutation produces significant disruption of osteoclast formation. The structures of RANK and RANKL-RANK complex and the biological data presented in the paper are essential for not only our understanding of the specific nature of the signaling mechanism and of disease-related mutations found in patients but also structure based drug design.

Assenberg R, Delmas O, Morin B, Graham SC, De Lamballerie X, Laubert C, Coutard B, Grimes JM, Neyts J, Owens RJ et al. 2010. Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription. Antiviral Res, 87 (2), pp. 149-161. | Show Abstract | Read more

Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication.

Nettleship JE, Assenberg R, Diprose JM, Rahman-Huq N, Owens RJ. 2010. Recent advances in the production of proteins in insect and mammalian cells for structural biology. J Struct Biol, 172 (1), pp. 55-65. | Show Abstract | Read more

The production of proteins in sufficient quantity and of appropriate quality is an essential pre-requisite for structural studies. Escherichia coli remains the dominant expression system in structural biology with nearly 90% of the structures in the Protein Data Bank (PDB) derived from proteins produced in this bacterial host. However, many mammalian and eukaryotic viral proteins require post-translation modification for proper folding and/or are part of large multimeric complexes. Therefore expression in higher eukaryotic cell lines from both invertebrate and vertebrate is required to produce these proteins. Although these systems are generally more time-consuming and expensive to use than bacteria, there have been improvements in technology that have streamlined the processes involved. For example, the use of multi-host vectors, i.e., containing promoters for not only E. coli but also mammalian and baculovirus expression in insect cells, enables target genes to be evaluated in both bacterial and higher eukaryotic hosts from a single vector. Culturing cells in micro-plate format allows screening of large numbers of vectors in parallel and is amenable to automation. The development of large-scale transient expression in mammalian cells offers a way of rapidly producing proteins with relatively high throughput. Strategies for selenomethionine-labelling (important for obtaining phase information in crystallography) and controlling glycosylation (important for reducing the chemical heterogeneity of glycoproteins) have also been reported for higher eukaryotic cell expression systems.

Ren J, Sainsbury S, Nettleship JE, Saunders NJ, Owens RJ. 2010. The crystal structure of NGO0477 from Neisseria gonorrhoeae reveals a novel protein fold incorporating a helix-turn-helix motif. Proteins, 78 (7), pp. 1798-1802. | Read more

Sainsbury S, Ren J, Nettleship JE, Saunders NJ, Stuart DI, Owens RJ. 2010. The structure of a reduced form of OxyR from Neisseria meningitidis. BMC Struct Biol, 10 (1), pp. 10. | Show Abstract | Read more

BACKGROUND: Survival of the human pathogen, Neisseria meningitidis, requires an effective response to oxidative stress resulting from the release of hydrogen peroxide by cells of the human immune system. In N. meningitidis, expression of catalase, which is responsible for detoxifying hydrogen peroxide, is controlled by OxyR, a redox responsive LysR-type regulator. OxyR responds directly to intracellular hydrogen peroxide through the reversible formation of a disulphide bond between C199 and C208 in the regulatory domain of the protein. RESULTS: We report the first crystal structure of the regulatory domain of an OxyR protein (NMB0173 from N. meningitidis) in the reduced state i.e. with cysteines at positions 199 and 208. The protein was crystallized under reducing conditions and the structure determined to a resolution of 2.4 A. The overall fold of the Neisseria OxyR shows a high degree of similarity to the structure of a C199S mutant OxyR from E. coli, which cannot form the redox sensitive disulphide. In the neisserial structure, C199 is located at the start of helix alpha3, separated by 18 A from C208, which is positioned between helices alpha3 and alpha4. In common with other LysR-type regulators, full length OxyR proteins are known to assemble into tetramers. Modelling of the full length neisserial OxyR as a tetramer indicated that C199 and C208 are located close to the dimer-dimer interface in the assembled tetramer. The formation of the C199-C208 disulphide may thus affect the quaternary structure of the protein. CONCLUSION: Given the high level of structural similarity between OxyR from N. meningitidis and E. coli, we conclude that the redox response mechanism is likely to be similar in both species, involving the reversible formation of a disulphide between C199-C208. Modelling suggests that disulphide formation would directly affect the interface between regulatory domains in an OxyR tetramer which in turn may lead to an alteration in the spacing/orientation of the DNA-binding domains and hence the interaction of OxyR with its DNA binding sites.

Bird LE, Ren J, Nettleship JE, Folkers GE, Owens RJ, Stammers DK. 2010. Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains. BMC Struct Biol, 10 (1), pp. 13. | Show Abstract | Read more

BACKGROUND: Zhx1 to 3 (zinc-fingers and homeoboxes) form a set of paralogous genes encoding multi-domain proteins. ZHX proteins consist of two zinc fingers followed by five homeodomains. ZHXs have biological roles in cell cycle control by acting as co-repressors of the transcriptional regulator Nuclear Factor Y. As part of a structural genomics project we have expressed single and multi-domain fragments of the different human ZHX genes for use in structure determination. RESULTS: A total of 30 single and multiple domain ZHX1-3 constructs selected from bioinformatics protocols were screened for soluble expression in E. coli using high throughput methodologies. Two homeodomains were crystallized leading to structures for ZHX1 HD4 and ZHX2 HD2. ZHX1 HD4, although closest matched to homeodomains from 'homez' and 'engrailed', showed structural differences, notably an additional C-terminal helix (helix V) which wrapped over helix I thereby making extensive contacts. Although ZHX2 HD2-3 was successfully expressed and purified, proteolysis occurred during crystallization yielding crystals of just HD2. The structure of ZHX2 HD2 showed an unusual open conformation with helix I undergoing 'domain-swapping' to form a homodimer. CONCLUSIONS: Although multiple-domain constructs of ZHX1 selected by bioinformatics studies could be expressed solubly, only single homeodomains yielded crystals. The crystal structure of ZHX1 HD4 showed additional hydrophobic interactions relative to many known homeodomains via extensive contacts formed by the novel C-terminal helix V with, in particular, helix I. Additionally, the replacement of some charged covariant residues (which are commonly observed to form salt bridges in non-homeotherms such as the Drosophila 'engrailed' homeodomain), by apolar residues further increases hydrophobic contacts within ZHX1 HD4, and potentially stability, relative to engrailed homeodomain. ZHX1 HD4 helix V points away from the normally observed DNA major groove binding site on homeodomains and thus would not obstruct the putative binding of nucleic acid. In contrast, for ZHX2 HD2 the observed altered conformation involving rearrangement of helix I, relative to the canonical homeodomain fold, disrupts the normal DNA binding site, although protein-protein binding is possible as observed in homodimer formation.

Hitchman RB, Possee RD, Siaterli E, Richards KS, Clayton AJ, Bird LE, Owens RJ, Carpentier DCJ, King FL, Danquah JO et al. 2010. Improved expression of secreted and membrane-targeted proteins in insect cells. Biotechnol Appl Biochem, 56 (3), pp. 85-93. | Show Abstract | Read more

Secretory and membrane-bound proteins are generally produced in lower amounts in insect cells compared with cytoplasmic and nuclear proteins. There may be many reasons for this, including degradation of recombinant proteins by proteases, competition for cellular resources between native and recombinant proteins, and physical blockage of the secretory pathways. In the present study, we describe the construction of a baculovirus in which chiA (chitinase) and cath (cathepsin) genes have been deleted and show improved recombinant protein expression using this vector. We confirmed the complete removal of both genes by PCR, restriction enzyme analysis and enzyme assays, and the modified virus DNA was shown to be stable in bacterial cells over multiple passages. A selection of recombinant genes were inserted into the double-deletion virus and their expression levels compared with recombinant viruses that had single or no gene deletions. In all instances, the double-deletion viruses showed greatly enhanced levels of protein production for both secreted and nuclear/cytoplasmic proteins. In summary, we have conclusively demonstrated the importance of this deletion vector for the high-level production of recombinant proteins.

Bowden TA, Aricescu AR, Nettleship JE, Siebold C, Rahman-Huq N, Owens RJ, Stuart DI, Jones EY. 2009. Structural Plasticity of Eph-Receptor A4 Facilitates Cross-Class Ephrin Signaling. Structure, 17 (12), pp. 1679. | Read more

Assenberg R, Mastrangelo E, Walter TS, Verma A, Milani M, Owens RJ, Stuart DI, Grimes JM, Mancini EJ. 2009. Crystal structure of a novel conformational state of the flavivirus NS3 protein: implications for polyprotein processing and viral replication. J Virol, 83 (24), pp. 12895-12906. | Show Abstract | Read more

The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-A-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed.

van Boxel GI, Stewart-Jones G, Holmes S, Sainsbury S, Shepherd D, Gillespie GMA, Harlos K, Stuart DI, Owens R, Jones EY. 2009. Some lessons from the systematic production and structural analysis of soluble (alpha)(beta) T-cell receptors. J Immunol Methods, 350 (1-2), pp. 14-21. | Show Abstract | Read more

T-cell receptors (TCRs) are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response. The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein. Here we report our systematic approach for generating soluble, (alpha)(beta)-TCRs, for X-ray crystallographic studies. By using small-scale expression screens, novel standardized quality control mechanisms and crystallization and imaging robots we were able to add significantly to the current TCR structural database. Our success in crystallizing both isolated TCRs and Major histocompatibility complex (MHC):TCR complexes has provided us with sufficient data to develop focused crystallization screens, which have proved generically useful for the crystallization of this family of proteins and complexes.

Bowden TA, Aricescu AR, Nettleship JE, Siebold C, Rahman-Huq N, Owens RJ, Stuart DI, Jones EY. 2009. Structural plasticity of eph receptor A4 facilitates cross-class ephrin signaling. Structure, 17 (10), pp. 1386-1397. | Show Abstract | Read more

The EphA4 tyrosine kinase cell surface receptor regulates an array of physiological processes and is the only currently known class A Eph receptor that binds both A and B class ephrins with high affinity. We have solved the crystal structure of the EphA4 ligand binding domain alone and in complex with (1) ephrinB2 and (2) ephrinA2. This set of structures shows that EphA4 has significant conformational plasticity in its ligand binding face. In vitro binding data demonstrate that it has a higher affinity for class A than class B ligands. Structural analyses, drawing on previously reported Eph receptor structures, show that EphA4 in isolation and in complex with ephrinA2 resembles other class A Eph receptors but on binding ephrinB2 assumes structural hallmarks of the class B Eph receptors. This interactive plasticity reveals EphA4 as a structural chameleon, able to adopt both A and B class Eph receptor conformations, and thus provides a molecular basis for EphA-type cross-class reactivity.

Hitchman RB, Possee RD, Crombie AT, Chambers A, Ho K, Siaterli E, Lissina O, Sternard H, Novy R, Loomis K et al. 2010. Genetic modification of a baculovirus vector for increased expression in insect cells. Cell Biol Toxicol, 26 (1), pp. 57-68. | Show Abstract | Read more

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.

Sainsbury S, Lane LA, Ren J, Gilbert RJ, Saunders NJ, Robinson CV, Stuart DI, Owens RJ. 2009. The structure of CrgA from Neisseria meningitidis reveals a new octameric assembly state for LysR transcriptional regulators. Nucleic Acids Res, 37 (14), pp. 4545-4558. | Show Abstract | Read more

LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes. The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial-host cell contact. Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings. Non-denaturing mass spectrometry analysis and analytical ultracentrifugation established that the octameric form of CrgA is the predominant species in solution in both the presence and absence of an oligonucleotide encompassing the CrgA-binding sequence. Furthermore, analysis of the isolated CrgA-DNA complex by mass spectrometry showed stabilization of a double octamer species upon DNA binding. Based on the observed structure and the mass spectrometry findings, a model is proposed in which a hexadecameric array of two CrgA oligomers binds to its DNA target site.

Wienk H, Lammers I, Hotze A, Wu J, Wechselberger RW, Owens R, Stammers DK, Stuart D, Kaptein R, Folkers GE. 2009. The tandem zinc-finger region of human ZHX adopts a novel C2H2 zinc finger structure with a C-terminal extension. Biochemistry, 48 (21), pp. 4431-4439. | Show Abstract | Read more

Binding of the nuclear factor-Y complex (NF-Y) to the inverted CCAAT-box interferes with transcription activation through nucleosome reorganization. The three homologous proteins forming the zinc-fingers and homeoboxes (ZHX) family interact with the activation domain of NF-Ya to repress transcription. Each ZHX-protein contains two generic C2H2 zinc-fingers (ZNF1 and ZNF2) followed by five homeodomains. Although the proteins have been related to the occurrence of certain cancers, the function and structure of the individual ZHX domains are still unknown. Here, we determined the structure of the tandem zinc-finger region of human ZHX1. Folding and secondary structure predictions combined with expression screening revealed that the C-terminal extension (E) to ZNF2 could form a single domain with the two hZHX1 zinc-fingers. We therefore decided to determine the solution structure of the zinc-fingers followed by this extension. We show that both zinc-fingers adopt canonical betabetaalpha-folds in which a zinc ion is coordinated by two cysteine and two histidine residues. The C-terminal extension to ZNF2 forms two beta-strands to make a beta-sheet with the beta-strands of this zinc-finger. The ZNF1 and ZNF2-E domains do not show evident contacts and their mutual orientation seems variable. The high degree of sequence conservation among ZHX family members permitted us to prepare homology models for ZHX2 and ZHX3, revealing distinct surface characteristics for each family member. Implications of these structural features for ZHX-functioning in transcription regulation are discussed.

Walter TS, Liu C, Huang P, Zhang S, Wedderburn LR, Gao B, Owens RJ, Stuart DI, Tang P, Ren J. 2009. Crystallization and preliminary X-ray analysis of mouse RANK and its complex with RANKL. Acta Crystallogr Sect F Struct Biol Cryst Commun, 65 (Pt 6), pp. 597-600. | Show Abstract | Read more

The interaction between the TNF-family molecule receptor activator of NF-kappaB ligand (RANKL) and its receptor RANK induces osteoclast formation, activation and survival in the process of bone remodelling. RANKL-RANK also plays critical roles in T-cell/dendritic cell communication and lymph-node formation and in a variety of pathologic conditions such as tumour-cell migration and bone metastasis. Both the ectodomain of mouse RANKL and the extracellular domain of mouse RANK have been cloned, expressed and purified. Crystals of RANK alone and of RANK in complex with RANKL have been obtained that are suitable for structure determination.

Nichols CE, Sainsbury S, Ren J, Walter TS, Verma A, Stammers DK, Saunders NJ, Owens RJ. 2009. The structure of NMB1585, a MarR-family regulator from Neisseria meningitidis. Acta Crystallogr Sect F Struct Biol Cryst Commun, 65 (Pt 3), pp. 204-209. | Show Abstract | Read more

The structure of the MarR-family transcription factor NMB1585 from Neisseria meningitidis has been solved using data extending to a resolution of 2.1 A. Overall, the dimeric structure resembles those of other MarR proteins, with each subunit comprising a winged helix-turn-helix (wHtH) domain connected to an alpha-helical dimerization domain. The spacing of the recognition helices of the wHtH domain indicates that NMB1585 is pre-configured for DNA binding, with a putative inducer pocket that is largely occluded by the side chains of two aromatic residues (Tyr29 and Trp53). NMB1585 was shown to bind to its own promoter region in a gel-shift assay, indicating that the protein acts as an auto-repressor.

Berrow NS, Alderton D, Owens RJ. 2009. The precise engineering of expression vectors using high-throughput In-Fusion PCR cloning. Methods Mol Biol, 498 pp. 75-90. | Show Abstract | Read more

In this chapter, protocols for the construction of expression vectors using In-Fusion PCR cloning are presented. The method enables vector and insert DNA sequences to be seamlessly joined in a ligation-independent reaction. This property of the In-Fusion process has been exploited in the design of a suite of multi-host compatible vectors for the expression of proteins with precisely engineered His-tags. Vector preparation, PCR amplification of the sequence to be cloned and the procedure for inserting the PCR product into the vector by In-Fusion are described.

Nettleship JE, Rahman-Huq N, Owens RJ. 2009. The production of glycoproteins by transient expression in Mammalian cells. Methods Mol Biol, 498 pp. 245-263. | Show Abstract | Read more

In this chapter, protocols for the growth and transfection of Human Embryonic Kidney (HEK) 293T cells for small scale expression screening and large scale protein production are described. Transient expression in mammalian cells offers a method of rapidly producing glycoproteins with a relatively high throughput. HEK 293T cells, in particular, can be transfected with high efficiency (> 50% cell expression) and are amenable to culture at multi-litre scale. Growing cells in micro-plate format allows screening of large numbers of vectors in parallel to prioritise those amenable to scale-up and purification for subsequent structural or functional studies. The glycoform of the expressed protein can be modified by treating cell cultures with kifunensine which inhibits glycan processing during protein synthesis. This results in the production of a chemically homogeneous glycoprotein with short mannose-rich sugar chains attached to the protein backbone. If required, these can be readily removed by endoglycosidase treatment.

Bowden TA, Aricescu AR, Nettleship JE, Siebold C, Rahman-Huq N, Owens RJ, Stuart DI, Jones EY. 2009. Structural Plasticity of Eph-Receptor A4 Facilitates Cross-Class Ephrin Signaling Structure, 17 (12), pp. 1679-1679. | Read more

Possee RD, Hitchman RB, Richards KS, Mann SG, Siaterli E, Nixon CP, Irving H, Assenberg R, Alderton D, Owens RJ, King LA. 2008. Generation of baculovirus vectors for the high-throughput production of proteins in insect cells. Biotechnol Bioeng, 101 (6), pp. 1115-1122. | Show Abstract | Read more

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to adapt to a multi-parallel process. We have developed a bacmid vector that does not require any form of selection pressure to separate recombinant virus from non-recombinant parental virus. The method relies on homologous recombination in insect cells between a transfer vector containing a gene to be expressed and a replication-deficient bacmid. The target gene replaces a bacterial replicon at the polyhedrin loci, simultaneously restoring a virus gene essential for replication. Therefore, only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses on automated platforms in a one-step procedure. Using this vector allowed us to automate the generation of multiple recombinant viruses with a robotic liquid handler and then rapidly screen infected insect cell supernatant for the presence of secreted proteins.

Graham SC, Assenberg R, Delmas O, Verma A, Gholami A, Talbi C, Owens RJ, Stuart DI, Grimes JM, Bourhy H. 2008. Rhabdovirus matrix protein structures reveal a novel mode of self-association. PLoS Pathog, 4 (12), pp. e1000251. | Show Abstract | Read more

The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins.

Nettleship JE, Ren J, Rahman N, Berrow NS, Hatherley D, Barclay AN, Owens RJ. 2008. A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells. Protein Expr Purif, 62 (1), pp. 83-89. | Show Abstract | Read more

We describe a pipeline for the rapid production of recombinant Fabs derived from mouse monoclonal antibodies suitable for use in structural studies. The pipeline is exemplified by the production of three Fabs derived from the monoclonal antibodies OX108 (anti-CD200 receptor), OX117 and OX119 (anti-SIRPgamma). Heavy and light chain variable domains were inserted into separate expression vectors containing resident constant regions using In-Fusion PCR cloning. Following transient co-expression in HEK 293T cells, secreted Fab fragments were purified by metal chelate chromatography and gel filtration using an automated procedure with yields of up to 4mg/L of cell culture. Following crystallization trials, diffracting crystals were obtained for the recombinant Fabs of OX108 and OX117, and their structures solved to 2.3A and 2.4A, respectively.

Meier C, Carter LG, Sainsbury S, Mancini EJ, Owens RJ, Stuart DI, Esnouf RM. 2008. The crystal structure of UMP kinase from Bacillus anthracis (BA1797) reveals an allosteric nucleotide-binding site. J Mol Biol, 381 (5), pp. 1098-1105. | Show Abstract | Read more

Uridine monophosphate (UMP) kinase is a conserved enzyme that catalyzes the ATP-driven conversion of uridylate monophosphate into uridylate diphosphate, an essential metabolic step. In prokaryotes, the enzyme exists as a homohexamer that is regulated by various metabolites. Whereas the enzymatic mechanism of UMP kinase (UK) is well-characterized, the molecular basis of its regulation remains poorly understood. Here we report the crystal structure of UK from Bacillus anthracis (BA1797) in complex with ATP at 2.82 A resolution. It reveals that the cofactor, in addition to binding in the active sites, also interacts with separate binding pockets located near the center of the hexameric structure. The existence of such an allosteric binding site had been predicted by biochemical studies, but it was not identified in previous crystal structures of prokaryotic UKs. We show that this putative allosteric pocket is conserved across different bacterial species, suggesting that it is a feature common to bacterial UKs, and we present a structural model for the allosteric regulation of this enzyme.

Sainsbury S, Ren J, Saunders NJ, Stuart DI, Owens RJ. 2008. Crystallization and preliminary X-ray analysis of CrgA, a LysR-type transcriptional regulator from pathogenic Neisseria meningitidis MC58. Acta Crystallogr Sect F Struct Biol Cryst Commun, 64 (Pt 9), pp. 797-801. | Show Abstract | Read more

Although LysR-type regulators (LTTRs) represent the largest family of transcriptional regulators in bacteria, the full-length structure of only one annotated LTTR (CbnR) has been deposited in the PDB. CrgA, a LTTR from pathogenic Neisseria meningitidis MC58, which is up-regulated upon bacterial cell contact with human epithelial cells, has been cloned, purified and crystallized. Crystals of full-length CrgA were obtained after buffer screening with a thermal shift assay and concentration with 0.2 M NDSB-256. Data were collected from two crystal forms of full-length CrgA belonging to space groups P2(1)2(1)2(1) and P2(1), diffracting to 3.0 and 3.8 A resolution and consistent with the presence of between six and ten and between ten and 20 copies of CrgA in the asymmetric unit, respectively. In addition, diffraction data were collected to 2.3 A resolution from the selenomethionine derivative of the regulatory domain of CrgA. The crystals belonged to space group P2(1) and contained two molecules in the asymmetric unit.

Graham SC, Bahar MW, Cooray S, Chen RA-J, Whalen DM, Abrescia NGA, Alderton D, Owens RJ, Stuart DI, Smith GL, Grimes JM. 2008. Vaccinia virus proteins A52 and B14 Share a Bcl-2-like fold but have evolved to inhibit NF-kappaB rather than apoptosis. PLoS Pathog, 4 (8), pp. e1000128. | Show Abstract | Read more

Vaccinia virus (VACV), the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-kappaB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 A and 2.7 A resolution, respectively. Strikingly, both these proteins adopt a Bcl-2-like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2-like proteins. Unlike cellular and viral Bcl-2-like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2-like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2-like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2-like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2-like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways.

Au K, Ren J, Walter TS, Harlos K, Nettleship JE, Owens RJ, Stuart DI, Esnouf RM. 2008. Structures of an alanine racemase from Bacillus anthracis (BA0252) in the presence and absence of (R)-1-aminoethylphosphonic acid (L-Ala-P). Acta Crystallogr Sect F Struct Biol Cryst Commun, 64 (Pt 5), pp. 327-333. | Show Abstract | Read more

Bacillus anthracis, the causative agent of anthrax, has been targeted by the Oxford Protein Production Facility to validate high-throughput protocols within the Structural Proteomics in Europe project. As part of this work, the structures of an alanine racemase (BA0252) in the presence and absence of the inhibitor (R)-1-aminoethylphosphonic acid (L-Ala-P) have determined by X-ray crystallography to resolutions of 2.1 and 1.47 A, respectively. Difficulties in crystallizing this protein were overcome by the use of reductive methylation. Alanine racemase has attracted much interest as a possible target for anti-anthrax drugs: not only is D-alanine a vital component of the bacterial cell wall, but recent studies also indicate that alanine racemase, which is accessible in the exosporium, plays a key role in inhibition of germination in B. anthracis. These structures confirm the binding mode of L-Ala-P but suggest an unexpected mechanism of inhibition of alanine racemase by this compound and could provide a basis for the design of improved alanine racemase inhibitors with potential as anti-anthrax therapies.

Coutard B, Gorbalenya AE, Snijder EJ, Leontovich AM, Poupon A, De Lamballerie X, Charrel R, Gould EA, Gunther S, Norder H et al. 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res, 78 (1), pp. 37-46. | Show Abstract | Read more

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

Assenberg R, Delmas O, Graham SC, Verma A, Berrow N, Stuart DI, Owens RJ, Bourhy H, Grimes JM. 2008. Expression, purification and crystallization of a lyssavirus matrix (M) protein. Acta Crystallogr Sect F Struct Biol Cryst Commun, 64 (Pt 4), pp. 258-262. | Show Abstract | Read more

The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 A resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 56.9-57.2, c = 187.9-188.6 A, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

Ren J, Nettleship JE, Sainsbury S, Saunders NJ, Owens RJ. 2008. Structure of the cold-shock domain protein from Neisseria meningitidis reveals a strand-exchanged dimer. Acta Crystallogr Sect F Struct Biol Cryst Commun, 64 (Pt 4), pp. 247-251. | Show Abstract | Read more

The structure of the cold-shock domain protein from Neisseria meningitidis has been solved to 2.6 A resolution and shown to comprise a dimer formed by the exchange of two beta-strands between protein monomers. The overall fold of the monomer closely resembles those of other bacterial cold-shock proteins. The neisserial protein behaved as a monomer in solution and was shown to bind to a hexathymidine oligonucleotide with a stoichiometry of 1:1 and a K(d) of 1.25 microM.

Structural Genomics Consortium, China Structural Genomics Consortium, Northeast Structural Genomics Consortium, Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, Knapp S et al. 2008. Protein production and purification. Nat Methods, 5 (2), pp. 135-146. | Show Abstract | Read more

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

Zaccai NR, Carter LG, Berrow NS, Sainsbury S, Nettleship JE, Walter TS, Harlos K, Owens RJ, Wilson KS, Stuart DI, Esnouf RM. 2008. Crystal structure of a 3-oxoacyl-(acylcarrier protein) reductase (BA3989) from Bacillus anthracis at 2.4-A resolution. Proteins, 70 (2), pp. 562-567. | Read more

Walter TS, Mancini EJ, Kadlec J, Graham SC, Assenberg R, Ren J, Sainsbury S, Owens RJ, Stuart DI, Grimes JM, Harlos K. 2008. Semi-automated microseeding of nanolitre crystallization experiments. Acta Crystallogr Sect F Struct Biol Cryst Commun, 64 (Pt 1), pp. 14-18. | Show Abstract | Read more

A simple semi-automated microseeding procedure for nanolitre crystallization experiments is described. Firstly, a microseed stock solution is made from microcrystals using a Teflon bead. A dilution series of this microseed stock is then prepared and dispensed as 100 nl droplets into 96-well crystallization plates, facilitating the incorporation of seeding into high-throughput crystallization pipelines. This basic microseeding procedure has been modified to include additive-screening and cross-seeding methods. Five examples in which these techniques have been used successfully are described.

Mancini EJ, Assenberg R, Verma A, Walter TS, Tuma R, Grimes JM, Owens RJ, Stuart DI. 2007. Structure of the Murray Valley encephalitis virus RNA helicase at 1.9 Angstrom resolution. Protein Sci, 16 (10), pp. 2294-2300. | Show Abstract | Read more

Murray Valley encephalitis virus (MVEV), a mosquito-borne flavivirus endemic to Australia, is closely related to Japanese encephalitis virus and West Nile virus. Nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains, whose activity is central to flavivirus replication and is therefore a possible target for anti-flaviviral compounds. Cloning, purification, and crystal structure determination to 1.9 Angstrom resolution of the NS3 helicase of MVEV and characterization of its enzymatic activity is reported. Comparison with the structures of helicases from related viruses supports a possible mechanism of ATP hydrolysis-driven strand separation.

Assenberg R, Ren J, Verma A, Walter TS, Alderton D, Hurrelbrink RJ, Fuller SD, Bressanelli S, Owens RJ, Stuart DI, Grimes JM. 2007. Crystal structure of the Murray Valley encephalitis virus NS5 methyltransferase domain in complex with cap analogues. J Gen Virol, 88 (Pt 8), pp. 2228-2236. | Show Abstract | Read more

We have determined the high resolution crystal structure of the methyltransferase domain of the NS5 polypeptide from the Murray Valley encephalitis virus. This domain is unusual in having both the N7 and 2'-O methyltransferase activity required for Cap 1 synthesis. We have also determined structures for complexes of this domain with nucleotides and cap analogues providing information on cap binding, based on which we suggest a model of how the sequential methylation of the N7 and 2'-O groups of the cap may be coordinated.

Ren J, Sainsbury S, Combs SE, Capper RG, Jordan PW, Berrow NS, Stammers DK, Saunders NJ, Owens RJ. 2007. The structure and transcriptional analysis of a global regulator from Neisseria meningitidis. J Biol Chem, 282 (19), pp. 14655-14664. | Show Abstract | Read more

Neisseria meningitidis, a causative agent of bacterial meningitis, has a relatively small repertoire of transcription factors, including NMB0573 (annotated AsnC), a member of the Lrp-AsnC family of regulators that are widely expressed in both Bacteria and Archaea. In the present study we show that NMB0573 binds to l-leucine and l-methionine and have solved the structure of the protein with and without bound amino acids. This has shown, for the first time that amino acid binding does not induce significant conformational changes in the structure of an AsnC/Lrp regulator although it does appear to stabilize the octameric assembly of the protein. Transcriptional profiling of wild-type and NMB0573 knock-out strains of N. meningitidis has shown that NMB0573 is associated with an adaptive response to nutrient poor conditions reflected in a reduction in major surface protein expression. On the basis of its structure and the transcriptional response, we propose that NMB0573 is a global regulator in Neisseria controlling responses to nutrient availability through indicators of general amino acid abundance: leucine and methionine.

Chang VT, Crispin M, Aricescu AR, Harvey DJ, Nettleship JE, Fennelly JA, Yu C, Boles KS, Evans EJ, Stuart DI et al. 2007. Glycoprotein structural genomics: solving the glycosylation problem. Structure, 15 (3), pp. 267-273. | Show Abstract | Read more

Glycoproteins present special problems for structural genomic analysis because they often require glycosylation in order to fold correctly, whereas their chemical and conformational heterogeneity generally inhibits crystallization. We show that the "glycosylation problem" can be solved by expressing glycoproteins transiently in mammalian cells in the presence of the N-glycosylation processing inhibitors, kifunensine or swainsonine. This allows the correct folding of the glycoproteins, but leaves them sensitive to enzymes, such as endoglycosidase H, that reduce the N-glycans to single residues, enhancing crystallization. Since the scalability of transient mammalian expression is now comparable to that of bacterial systems, this approach should relieve one of the major bottlenecks in structural genomic analysis.

Meier C, Carter LG, Winter G, Owens RJ, Stuart DI, Esnouf RM. 2007. Structure of 5-formyltetrahydrofolate cyclo-ligase from Bacillus anthracis (BA4489). Acta Crystallogr Sect F Struct Biol Cryst Commun, 63 (Pt 3), pp. 168-172. | Show Abstract | Read more

Bacillus anthracis is a spore-forming bacterium and the causative agent of the disease anthrax. The Oxford Protein Production Facility has been targeting proteins from B. anthracis in order to develop high-throughput technologies within the Structural Proteomics in Europe project. As part of this work, the structure of 5-formyltetrahydrofolate cyclo-ligase (BA4489) has been determined by X-ray crystallography to 1.6 A resolution. The structure, solved in complex with magnesium-ion-bound ADP and phosphate, gives a detailed picture of the proposed catalytic mechanism of the enzyme. Chemical differences from other cyclo-ligase structures close to the active site that could be exploited to design specific inhibitors are also highlighted.

Nettleship JE, Aplin R, Aricescu AR, Evans EJ, Davis SJ, Crispin M, Owens RJ. 2007. Analysis of variable N-glycosylation site occupancy in glycoproteins by liquid chromatography electrospray ionization mass spectrometry. Anal Biochem, 361 (1), pp. 149-151. | Read more

Brookings D, Davenport RJ, Davis J, Galvin FCA, Lloyd S, Mack SR, Owens R, Sabin V, Wynn J. 2007. Novel nucleotide triphosphates as potent P2Y2 agonists. Bioorg Med Chem Lett, 17 (2), pp. 562-565. | Show Abstract | Read more

The synthesis and P2Y2 activities of a novel series of nucleoside triphosphates are described. Many of these compounds were potent agonists of the P2Y2 receptor.

Davenport RJ, Diaz P, Galvin FCA, Lloyd S, Mack SR, Owens R, Sabin V, Wynn J. 2007. Novel nucleotide triphosphates as potent P2Y2 agonists with enhanced stability over UTP. Bioorg Med Chem Lett, 17 (2), pp. 558-561. | Show Abstract | Read more

The synthesis of a series of novel C-linked nucleotide triphosphates is reported. These exhibit excellent agonist potency and selectivity for the P2Y2 receptor with a number of examples having EC50 values below 10 nM. Representative compounds from the N-linked and C-linked series showed enhanced metabolic stability compared with that of the natural ligand UTP.

Berrow NS, Alderton D, Sainsbury S, Nettleship J, Assenberg R, Rahman N, Stuart DI, Owens RJ. 2007. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res, 35 (6), pp. e45. | Show Abstract | Read more

This article describes the construction of a set of versatile expression vectors based on the In-Fusion cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His(6)-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.

Fogg MJ, Alzari P, Bahar M, Bertini I, Betton J-M, Burmeister WP, Cambillau C, Canard B, Carrondo MA, Coll M et al. 2006. Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens (vol 62, pg 1196, 2006) ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, 62 (12), pp. 1571-1571. | Read more

Walter TS, Meier C, Assenberg R, Au K-F, Ren J, Verma A, Nettleship JE, Owens RJ, Stuart DI, Grimes JM. 2006. Lysine methylation as a routine rescue strategy for protein crystallization. Structure, 14 (11), pp. 1617-1622. | Show Abstract | Read more

Crystallization remains a critical step in X-ray structure determination. Because it is not generally possible to rationally predict crystallization conditions, commercial screens have been developed which sample a wide range of crystallization space. While this approach has proved successful in many cases, a significant number of proteins fail to crystallize despite being soluble and monodispersed. It is established that chemical modification can facilitate the crystallization of otherwise intractable proteins. Here we describe a method for the reductive methylation of lysine residues which is simple, inexpensive, and efficient, and report on its application to ten proteins. We describe the effect of methylation on the physico-chemical properties of these proteins, and show that it led to diffraction-quality crystals from four proteins and structures for three that had hitherto proved refractory to crystallization. The method is suited to both low- and high-throughput laboratories.

Fogg MJ, Alzari P, Bahar M, Bertini I, Betton JM, Burmeister WP, Cambillau C, Canard B, Corrondo MA, Coll M et al. 2006. Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1196-1207. | Show Abstract | Read more

The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.

Au K, Berrow NS, Blagova E, Boucher IW, Boyle MP, Brannigan JA, Carter LG, Dierks T, Folkers G, Grenha R et al. 2006. Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1267-1275. | Show Abstract | Read more

A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.

Banci L, Bertini I, Cusack S, de Jong RN, Heinemann U, Jones EY, Kozielski F, Maskos K, Messerschmidt A, Owens R et al. 2006. First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1208-1217. | Show Abstract | Read more

The EC 'Structural Proteomics In Europe' contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, approximately 170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

Berrow NS, Büssow K, Coutard B, Diprose J, Ekberg M, Folkers GE, Levy N, Lieu V, Owens RJ, Peleg Y et al. 2006. Recombinant protein expression and solubility screening in Escherichia coli: a comparative study. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1218-1226. | Show Abstract | Read more

Producing soluble proteins in Escherichia coli is still a major bottleneck for structural proteomics. Therefore, screening for soluble expression on a small scale is an attractive way of identifying constructs that are likely to be amenable to structural analysis. A variety of expression-screening methods have been developed within the Structural Proteomics In Europe (SPINE) consortium and to assist the further refinement of such approaches, eight laboratories participating in the network have benchmarked their protocols. For this study, the solubility profiles of a common set of 96 His(6)-tagged proteins were assessed by expression screening in E. coli. The level of soluble expression for each target was scored according to estimated protein yield. By reference to a subset of the proteins, it is demonstrated that the small-scale result can provide a useful indicator of the amount of soluble protein likely to be produced on a large scale (i.e. sufficient for structural studies). In general, there was agreement between the different groups as to which targets were not soluble and which were the most soluble. However, for a large number of the targets there were wide discrepancies in the results reported from the different screening methods, which is correlated with variations in the procedures and the range of parameters explored. Given finite resources, it appears that the question of how to most effectively explore ;expression space' is similar to several other multi-parameter problems faced by crystallographers, such as crystallization.

Alzari PM, Berglund H, Berrow NS, Blagova E, Busso D, Cambillau C, Campanacci V, Christodoulou E, Eiler S, Fogg MJ et al. 2006. Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1103-1113. | Show Abstract | Read more

The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation.

Geerlof A, Brown J, Coutard B, Egloff MP, Enguita FJ, Fogg MJ, Gilbert RJC, Groves MR, Haouz A, Nettleship JE et al. 2006. The impact of protein characterization in structural proteomics. Acta Crystallogr D Biol Crystallogr, 62 (Pt 10), pp. 1125-1136. | Show Abstract | Read more

Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein-protein and protein-ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given.

Nichols CE, Sainsbury S, Berrow NS, Alderton D, Saunders NJ, Stammers DK, Owens RJ. 2006. Structure of the PII signal transduction protein of Neisseria meningitidis at 1.85 A resolution. Acta Crystallogr Sect F Struct Biol Cryst Commun, 62 (Pt 6), pp. 494-497. | Show Abstract | Read more

The P(II) signal transduction proteins GlnB and GlnK are implicated in the regulation of nitrogen assimilation in Escherichia coli and other enteric bacteria. P(II)-like proteins are widely distributed in bacteria, archaea and plants. In contrast to other bacteria, Neisseria are limited to a single P(II) protein (NMB 1995), which shows a high level of sequence identity to GlnB and GlnK from Escherichia coli (73 and 62%, respectively). The structure of the P(II) protein from N. meningitidis (serotype B) has been solved by molecular replacement to a resolution of 1.85 A. Comparison of the structure with those of other P(II) proteins shows that the overall fold is tightly conserved across the whole population of related proteins, in particular the positions of the residues implicated in ATP binding. It is proposed that the Neisseria P(II) protein shares functions with GlnB/GlnK of enteric bacteria.

Fogg MJ, Alzari P, Bahar M, Bertini I, Betton JM, Burmeister WP, Cambillau C, Canard B, Carrondo MA, Coll M et al. 2006. Erratum: Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens (Acta Crystallographica Section D: Biological Crystallography (2006) D62 (1196-1207)) Acta Crystallographica Section D: Biological Crystallography, 62 (12), pp. 1571. | Read more

Siebold C, Berrow N, Walter TS, Harlos K, Owens RJ, Stuart DI, Terman JR, Kolodkin AL, Pasterkamp RJ, Jones EY. 2005. High-resolution structure of the catalytic region of MICAL (molecule interacting with CasL), a multidomain flavoenzyme-signaling molecule. Proc Natl Acad Sci U S A, 102 (46), pp. 16836-16841. | Show Abstract | Read more

Semaphorins are extracellular cell guidance cues that govern cytoskeletal dynamics during neuronal and vascular development. MICAL (molecule interacting with CasL) is a multidomain cytosolic protein with a putative flavoprotein monooxygenase (MO) region required for semaphorin-plexin repulsive axon guidance. Here, we report the 1.45-A resolution crystal structure of the FAD-containing MO domain of mouse MICAL-1 (residues 1-489). The topology most closely resembles that of the NADPH-dependent flavoenzyme p-hydroxybenzoate hydroxylase (PHBH). Comparison of structures before and after reaction with NADPH reveals that, as in PHBH, the flavin ring can switch between two discrete positions. In contrast with other MOs, this conformational switch is coupled with the opening of a channel to the active site, suggestive of a protein substrate. In support of this hypothesis, distinctive structural features highlight putative protein-binding sites in suitable proximity to the active site entrance. The unusual juxtaposition of this N-terminal MO (hydroxylase) activity with the characteristics of a multiprotein-binding scaffold exhibited by the C-terminal portion of the MICALs represents a unique combination of functionality to mediate signaling.

Ren J, Sainsbury S, Berrow NS, Alderton D, Nettleship JE, Stammers DK, Saunders NJ, Owens RJ. 2005. Crystal structure of nitrogen regulatory protein IIANtr from Neisseria meningitidis. BMC Struct Biol, 5 (1), pp. 13. | Show Abstract | Read more

BACKGROUND: The NMB0736 gene of Neisseria meningitidis serogroup B strain MC58 encodes the putative nitrogen regulatory protein, IIANtr (abbreviated to NM-IIANtr). The homologous protein present in Escherichia coli is implicated in the control of nitrogen assimilation. As part of a structural proteomics approach to the study of pathogenic Neisseria spp., we have selected this protein for structure determination by X-ray crystallography. RESULTS: The NM-IIANtr was over-expressed in E. coli and was shown to be partially mono-phosphorylated, as assessed by mass spectrometry of the purified protein. Crystals of un-phosphorylated protein were obtained and diffraction data collected to 2.5 A resolution. The structure of NM-IIANtr was solved by molecular replacement using the coordinates of the E. coli nitrogen regulatory protein IIAntr [PDB: 1A6J] as the starting model. The overall fold of the Neisseria enzyme shows a high degree of similarity to the IIANtr from E. coli, and the position of the phosphoryl acceptor histidine residue (H67) is conserved. The orientation of an adjacent arginine residue (R69) suggests that it may also be involved in coordinating the phosphate group. Comparison of the structure with that of E. coli IIAmtl complexed with HPr [PDB: 1J6T] indicates that NM-IIANtr binds in a similar way to the HPr-like enzyme in Neisseria. CONCLUSION: The structure of NM-IIANtr confirms its assignment as a homologue of the IIANtr proteins found in a range of other Gram-negative bacteria. We conclude that the NM- IIANtr protein functions as part of a phosphorylation cascade which, in contrast to E. coli, shares the upstream phosphotransfer protein with the sugar uptake phosphoenolpyruvate:sugar phosphotransferase system (PTS), but in common with E. coli has a distinct downstream effector mechanism.

Walter TS, Diprose JM, Mayo CJ, Siebold C, Pickford MG, Carter L, Sutton GC, Berrow NS, Brown J, Berry IM et al. 2005. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr D Biol Crystallogr, 61 (Pt 6), pp. 651-657. | Show Abstract | Read more

Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.

Nettleship JE, Walter TS, Aplin R, Stammers DK, Owens RJ. 2005. Sample preparation and mass-spectrometric characterization of crystal-derived protein samples. Acta Crystallogr D Biol Crystallogr, 61 (Pt 5), pp. 643-645. | Show Abstract | Read more

Mass spectrometry is often used to ascertain the accurate mass of purified protein samples prior to crystallization screening. However, in many cases data regarding the form of the protein crystallizing can also be useful, as this may differ from the original sample. Development of a simple method for the preparation and mass spectrometry of crystal-derived protein samples is described. The method is exemplified by the determination of the phosphorylation state of protein in a crystal derived from a mixture of phosphorylated and unphosphorylated protein.

Mayo CJ, Diprose JM, Walter TS, Berry IM, Wilson J, Owens RJ, Jones EY, Harlos K, Stuart DI, Esnouf RM. 2005. Benefits of automated crystallization plate tracking, imaging, and analysis. Structure, 13 (2), pp. 175-182. | Show Abstract | Read more

We describe the design of a database and software for managing and organizing protein crystallization data. We also outline the considerations behind the design of a fast web interface linking protein production data, crystallization images, and automated image analysis. The database and associated interfaces underpin the Oxford Protein Production Facility (OPPF) crystallization laboratory, collecting, in a routine and automatic manner, up to 100,000 images per day. Over 17 million separate images are currently held in this database. We discuss the substantial scientific benefits automated tracking, imaging, and analysis of crystallizations offers to the structural biologist: analysis of the time course of the trial and easy analysis of trials with related crystallization conditions. Features of this system address requirements common to many crystallographic laboratories that are currently setting up (semi-)automated crystallization imaging systems.

Ren J, Sainsbury S, Berrow NS, Alderton D, Nettleship JE, Stammers DK, Saunders NJ, Owens RJ. 2005. Crystal structure of nitrogen regulatory protein IIA<sup>Ntr</sup> from Neisseria meningitidis BMC Structural Biology, 5 | Show Abstract | Read more

Background: The NMB0736 gene of Neisseria meningitidis serogroup B strain MC58 encodes the putative nitrogen regulatory protein, IIANtr (abbreviated to NM-IIANtr). The homologous protein present in Escherichia coli is implicated in the control of nitrogen assimilation. As part of a structural proteomics approach to the study of pathogenic Neisseria spp., we have selected this protein for structure determination by X-ray crystallography. Results: The NM-IIANtr was over-expressed in E. coli and was shown to be partially monophosphorylated, as assessed by mass spectrometry of the purified protein. Crystals of un-phosphorylated protein were obtained and diffraction data collected to 2.5 Å resolution. The structure of NM-IIANtr was solved by molecular replacement using the coordinates of the E. coli nitrogen regulatory protein IIAntr [PDB: 1A6J] as the starting model. The overall fold of the Neisseria enzyme shows a high degree of similarity to the IIANtr from E. coli, and the position of the phosphoryl acceptor histidine residue (H67) is conserved. The orientation of an adjacent arginine residue (R69) suggests that it may also be involved in coordinating the phosphate group. Comparison of the structure with that of E. coli IIAmtl complexed with HPr [PDB: 1J6T] indicates that NM-IIANtr binds in a similar way to the HPr-like enzyme in Neisseria. Conclusion: The structure of NM-IIANtr confirms its assignment as a homologue of the IIANtr proteins found in a range of other Gram-negative bacteria. We conclude that the NM- IIANtr protein functions as part of a phosphorylation cascade which, in contrast to E. coli, shares the upstream phosphotransfer protein with the sugar uptake phosphoenolpyruvate:sugar phosphotransferase system (PTS), but in common with E. coli has a distinct downstream effector mechanism. © 2005 Ren et al; licensee BioMed Central Ltd.

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Sainsbury S, Berrow NS, Alderton D, Nettleship JE, Stammers DK, Owens RJ, Ren J, Saunders NJ. 2005. Crystal structure of nitrogen regulatory protein IIA from Neisseria meningitidis BMC Structural Biology, 5 | Show Abstract | Read more

Background: The NMB0736 gene of Neisseria meningitidis serogroup B strain MC58 encodes the putative nitrogen regulatory protein, IIA (abbreviated to NM-IIA). The homologous protein present in Escherichia coli is implicated in the control of nitrogen assimilation. As part of a structural proteomics approach to the study of pathogenic Neisseria spp., we have selected this protein for structure determination by X-ray crystallography. Results: The NM-IIA was over-expressed in E. coli and was shown to be partially monophosphorylated, as assessed by mass spectrometry of the purified protein. Crystals of un-phosphorylated protein were obtained and diffraction data collected to 2.5 Å resolution. The structure of NM-IIA was solved by molecular replacement using the coordinates of the E. coli nitrogen regulatory protein IIA [PDB: 1A6J] as the starting model. The overall fold of the Neisseria enzyme shows a high degree of similarity to the IIA from E. coli, and the position of the phosphoryl acceptor histidine residue (H67) is conserved. The orientation of an adjacent arginine residue (R69) suggests that it may also be involved in coordinating the phosphate group. Comparison of the structure with that of E. coli IIA complexed with HPr [PDB: 1J6T] indicates that NM-IIA binds in a similar way to the HPr-like enzyme in Neisseria. Conclusion: The structure of NM-IIA confirms its assignment as a homologue of the IIA proteins found in a range of other Gram-negative bacteria. We conclude that the NM- IIA protein functions as part of a phosphorylation cascade which, in contrast to E. coli, shares the upstream phosphotransfer protein with the sugar uptake phosphoenolpyruvate:sugar phosphotransferase system (PTS), but in common with E. coli has a distinct downstream effector mechanism. © 2005 Ren et al; licensee BioMed Central Ltd.

Sutton G, Fry E, Carter L, Sainsbury S, Walter T, Nettleship J, Berrow N, Owens R, Gilbert R, Davidson A et al. 2004. The nsp9 replicase protein of SARS-coronavirus, structure and functional insights. Structure, 12 (2), pp. 341-353. | Show Abstract | Read more

As part of a high-throughput structural analysis of SARS-coronavirus (SARS-CoV) proteins, we have solved the structure of the non-structural protein 9 (nsp9). This protein, encoded by ORF1a, has no designated function but is most likely involved with viral RNA synthesis. The protein comprises a single beta-barrel with a fold previously unseen in single domain proteins. The fold superficially resembles an OB-fold with a C-terminal extension and is related to both of the two subdomains of the SARS-CoV 3C-like protease (which belongs to the serine protease superfamily). nsp9 has, presumably, evolved from a protease. The crystal structure suggests that the protein is dimeric. This is confirmed by analytical ultracentrifugation and dynamic light scattering. We show that nsp9 binds RNA and interacts with nsp8, activities that may be essential for its function(s).

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Brown J, Walter TS, Carter L, Abrescia NGA, Aricescu AR, Batuwangala TD, Bird LE, Brown N, Chamberlain PP, Davis SJ et al. 2003. A procedure for setting up high-throughput nanolitre crystallization experiments. II. Crystallization results JOURNAL OF APPLIED CRYSTALLOGRAPHY, 36 (2), pp. 315-318. | Show Abstract | Read more

<jats:p>An initial tranche of results from day-to-day use of a robotic system for setting up 100 nl-scale vapour-diffusion sitting-drop protein crystallizations has been surveyed. The database of over 50 unrelated samples represents a snapshot of projects currently at the stage of crystallization trials in Oxford research groups and as such encompasses a broad range of proteins. The results indicate that the nanolitre-scale methodology consistently identifies more crystallization conditions than traditional hand-pipetting-style methods; however, in a number of cases successful scale-up is then problematic. Crystals grown in the initial 100 nl-scale drops have in the majority of cases allowed useful characterization of X-ray diffraction, either in-house or at synchrotron beamlines. For a significant number of projects, full X-ray diffraction data sets have been collected to 3 Å resolution or better (either in-house or at the synchrotron) from crystals grown at the 100 nl scale. To date, five structures have been determined by molecular replacement directly from such data and a further three from scale-up of conditions established at the nanolitre scale.</jats:p>

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Walter TS, Diprose J, Brown J, Pickford M, Owens RJ, Stuart DI, Harlos K. 2003. A procedure for setting up high-throughput nanolitre crystallization experiments. I. Protocol design and validation JOURNAL OF APPLIED CRYSTALLOGRAPHY, 36 (2), pp. 308-314. | Show Abstract | Read more

<jats:p>A protocol for setting up nanolitre sitting-drop vapour-diffusion experiments is reported. The procedure uses standard crystallization screening kits and 96-well crystallization plates. Reservoir solutions are transferred from 96-deep-well blocks to crystallization plates in a single step with a Robbins-Hydra pipettor. Nanolitre droplets of protein as well as reservoir solution are dispensed by a Cartesian pipetting instrument. Experiments have been carried out to characterize the performance of this instrument. Adaptations to the Cartesian, which include an anti-evaporation cover plate, are described and tested. The protocol was designed for a high-throughput facility, but can be used in any standard crystallography laboratory.</jats:p>

Wan T, Beavil RL, Fabiane SM, Beavil AJ, Sohi MK, Keown M, Young RJ, Henry AJ, Owens RJ, Gould HJ, Sutton BJ. 2002. The crystal structure of IgE Fc reveals an asymmetrically bent conformation. Nat Immunol, 3 (7), pp. 681-686. | Show Abstract | Read more

The distinguishing structural feature of immunoglobulin E (IgE), the antibody responsible for allergic hypersensitivity, is the C epsilon 2 domain pair that replaces the hinge region of IgG. The crystal structure of the IgE Fc (constant fragment) at a 2.6-A resolution has revealed these domains. They display a distinctive, disulfide-linked Ig domain interface and are folded back asymmetrically onto the C epsilon 3 and C epsilon 4 domains, which causes an acute bend in the IgE molecule. The structure implies that a substantial conformational change involving C epsilon 2 must accompany binding to the mast cell receptor Fc epsilon RI. This may be the basis of the exceptionally slow dissociation rate of the IgE-Fc epsilon RI complex and, thus, of the ability of IgE to cause persistent allergic sensitization of mast cells.

Holland K, Gozzard N, Owens R, Foulkes R. 1999. Fc epsilon RI immunoconjugate inhibits IgE-mediated passive cutaneous anaphylaxis (PCA) in the rat BRITISH JOURNAL OF PHARMACOLOGY, 128 pp. U30-U30.

Allen RA, Merriman MW, Perry MJ, Owens RJ. 1999. Development of a recombinant cell-based system for the characterisation of phosphodiesterase 4 isoforms and evaluation of inhibitors. Biochem Pharmacol, 57 (12), pp. 1375-1382. | Show Abstract | Read more

We described the development of a recombinant cell-based system for the characterisation of phosphodiesterase (PDE) 4 isoforms and the evaluation of inhibitors. The Chinese hamster ovary (CHO) cell, which was found to have a low endogenous PDE4 background and no beta-adrenergic receptors (beta-AR), was transiently transfected with beta-AR and various PDE4 isoforms which were expressed as functionally coupled molecules. From correlations of elevation of adenosine 3',5'-cyclic monophosphate in situ and the inhibition of catalytic activity in vitro with the various PDE4 isoforms, it was apparent that PDE4A4, 4B2, 4C2, 4D2, and 4D3 all adopted a high-affinity binding conformation (i.e. expressed the high-affinity rolipram binding site) in the CHO cell, whereas PDE4A330 was expressed in a low-affinity conformation in situ. This gives the opportunity of using this system to screen and optimise inhibitors against a low-affinity conformation of PDE4 in situ and use a high-affinity conformation of PDE4 as a counterscreen, as inhibitor activity against this conformer has been linked with undesirable side effects. This system could also be utilised to screen inhibitors against various PDE4 isoforms in isolation against a low endogenous PDE background in situ for isoform-selective inhibitors.

Perry MJ, O'Connell J, Walker C, Crabbe T, Baldock D, Russell A, Lumb S, Huang Z, Howat D, Allen R et al. 1998. CDP840: a novel inhibitor of PDE-4. Cell Biochem Biophys, 29 (1-2), pp. 113-132. | Show Abstract | Read more

We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-¿3-cyclopentyloxy-4-methoxyphenyl¿-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC50 2-30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC50 > 100 microM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes, indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were confirmed by the use of a PDE-4A variant, PDE-4A330-886, which rolipram inhibited with low affinity (IC50 = 1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC50 = 3.9 nM), which was in good agreement with the Kd of 4.8 nM obtained from [3H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [3H]-rolipram binding to PDE-4A, B, C, and D with similar Kd app (7-19 nM and 3-5 nM, respectively). Thus, the activity of CDP840 at the [3H]-rolipram binding site was in agreement with the inhibitor's activity at the catalytic site. However, rolipram was approximately 100-fold more potent than CDP840 at inhibiting the binding of [3H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4 isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr).

Cook JP, Henry AJ, McDonnell JM, Owens RJ, Sutton BJ, Gould HJ. 1997. Identification of contact residues in the IgE binding site of human FcepsilonRIalpha. Biochemistry, 36 (50), pp. 15579-15588. | Show Abstract | Read more

The high-affinity receptor for immunoglobulin E (IgE), FcepsilonRI, is an alphabetagamma2 tetramer found on mast cells, basophils, and several other types of immune effector cells. The interaction of IgE with the alpha-subunit of FcepsilonRI is central to the pathogenesis of allergy. Detailed knowledge of the mode of interaction of FcepsilonRI with IgE may facilitate the development of inhibitors for general use in the treatment of allergic disease. To this end we have performed site-directed mutagenesis on a soluble form of the FcepsilonRI alpha-chain (sFcepsilonRIalpha). The effects of four mutations in the second immunoglobulin-like domain of sFcepsilonRIalpha upon the kinetics of binding to IgE and fragments of IgE have been analyzed using surface plasmon resonance. As described in the preceding paper of this issue [Henry, A. J., et al. (1997) Biochemistry 36, 15568-15578], biphasic binding kinetics was observed. Two of the mutations had significant effects on binding: K117D reduced the affinity of sFcepsilonRIalpha for IgE by a factor of 30, while D159K increased the affinity for IgE by a factor of 7, both principally through changes in the rates of dissociation of the slower phase of the interaction. Circular dichroism spectra of sFcepsilonRIalpha incorporating either of these mutations were indistinguishable from those of wild-type sFcepsilonRIalpha, demonstrating that the native conformation had not been disrupted. Our results, together with those from site-directed mutagenesis on fragments of IgE presented in the accompanying paper, define the contact surfaces in the IgE:sFcepsilonRIalpha complex.

Owens RJ, Lumb S, Rees-Milton K, Russell A, Baldock D, Lang V, Crabbe T, Ballesteros M, Perry MJ. 1997. Molecular cloning and expression of a human phosphodiesterase 4C. Cell Signal, 9 (8), pp. 575-585. | Show Abstract | Read more

A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5' end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5' splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the Km for cAMP of the enzyme produced in COS cells was 0.6 microM compared to 2.6 microM for the yeast 4C activity. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC50 of 23 nM compared to 1648 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [3H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.

Huston E, Lumb S, Russell A, Catterall C, Ross AH, Steele MR, Bolger GB, Perry MJ, Owens RJ, Houslay MD. 1997. Molecular cloning and transient expression in COS7 cells of a novel human PDE4B cAMP-specific phosphodiesterase, HSPDE4B3. Biochem J, 328 ( Pt 2) (2), pp. 549-558. | Show Abstract | Read more

5'-Rapid amplification of cDNA ends, done on poly(A)+ RNA from human U87 cells, was used to identify 420 bp of novel 5' sequence of a PDE4B cAMP-specific phosphodiesterase (PDE). This identified an open reading frame encoding a putative 721-residue 'long-form' PDE4B splice variant, which we term HSPDE4B3. HSPDE4B3 differs from the two known PDE4B forms by virtue of its unique 79-residue N-terminal region, compared with the unique N-terminal regions of 94 and 39 residues found in HSPDE4B1 and HSPDE4B2 respectively. In transfected COS7 cells the two long forms, HSPDE4B1 and HSPDE4B3, had molecular masses of approx. 104 and approx. 103 kDa respectively. Expressed in COS-7 cells, the three HSPDE4B isoforms were found in the high-speed supernatant (cytosol) fraction as well as both the high-speed pellet (P2) and low-speed pellet (P1) fractions. All isoforms showed similar Km values for cAMP hydrolysis (1.5-2.6 microM). The maximal activities of the soluble cytosolic activity of the two long forms were very similar, whereas that of the short form, HSPDE4B2, was approx. 4-fold higher. Particulate-associated HSPDE4B1 and HSPDE4B2 were less active (approx. 40%) than their cytosol forms, whereas particulate HSPDE4B3 was similar in activity to its cytosolic form. Particulate and cytosolic forms of HSPDE4B1 and HSPDE4B3 were similarly inhibited by rolipram {4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone}, the selective inhibitor of PDE4 (IC50 0.05-0.1 microM), whereas particulate-associated HSPDE4B2 was profoundly (approx. 10-fold) more sensitive (IC50 0.02 microM) to rolipram inhibition than its cytosolic form (IC50 0.2 microM). The various particulate-associated HSPDE4B isoforms showed very different susceptibilities to solubilization with the detergent Triton X-100 and high NaCl concentration. A novel cDNA, called pRPDE74, was obtained by screening a rat olfactory lobe cDNA library. This contained an open reading frame encoding a 721-residue protein that showed approx. 96% amino acid identity with HSPDE4B3 and is proposed to reflect the rat homologue of this human enzyme and is thus called RNPDE4B3. Alternative splicing of mRNA generated from both the human and rat PDE4B genes produces long and short splice variants that have unique N-terminal splice regions. It is suggested that these alternatively spliced regions determine changes in the maximal catalytic activity of the isoforms, their susceptibility to inhibition by rolipram and mode of interaction with particulate fractions.

Owens RJ, Catterall C, Batty D, Jappy J, Russell A, Smith B, O'Connell J, Perry MJ. 1997. Human phosphodiesterase 4A: characterization of full-length and truncated enzymes expressed in COS cells. Biochem J, 326 ( Pt 1) (1), pp. 53-60. | Show Abstract | Read more

The type 4 phosphodiesterase (PDE) family comprises four enzymes (4A, 4B, 4C and 4D) that are characterized by their specificity for cAMP and selective inhibition by the anti-depressant drug rolipram (4-[3-(cyclopentoxyl)-4-methoxyphenyl]2-pyrrolidone). In common with other PDEs, they consist of a central conserved domain associated with catalytic activity in addition to two N-terminal upstream conserved regions (UCR1 and UCR2) that are unique to the type 4 enzymes. We have isolated a 2 kb cDNA encoding a full-length type 4A PDE{HSPDE4A4B[Bolger, Michaeli, Martins, St.John, Steiner, Rodgers, Riggs, Wigler and Ferguson (1993) Mol. Cell. Biol. 13, 6558-6571]} from a human frontal cortex cDNA library. Northern blot analysis showed that the major PDE4A mRNA of 4.5 kb was widely distributed in different human tissues. The recombinant PDE4A expressed in COS cells had a molecular mass of approx. 117 kDa as revealed by SDS/PAGE/Western blotting with a PDE4A-specific antibody and was specific for cAMP with a Km of 4.8 microM. The enzyme activity was potently inhibited by R-rolipram (IC50 204 nM) and showed a 2.7-fold stereoselectivity over the S enantiomer. Analysis of the kinetics of inhibition indicated that R-rolipram did not behave as a simple competitive inhibitor. Dixon replots suggested that there was more than one mode of interaction consistent with the detection in the enzyme of a high-affinity binding site for R-rolipram with a Kd of 2.3 nM. Truncation of the PDE4A enzyme by deletion mutagenesis showed that neither of the UCRs was required for catalytic activity and identified an approx. 71 kDa core enzyme with a K(m) for cAMP of 3.3 microM. In contrast with the full-length PDE4A, R-rolipram behaved as a simple competitive inhibitor of this form of the enzyme with decreased potency (IC50 1022 nM) and no stereoselectivity. In addition, no high-affinity rolipram-binding site was detected in the truncated enzyme, indicating that this interaction involves sequences upstream of the catalytic domain of the enzyme.

Owens R, Ball E, Ganesh R, Nesbitt A, Brown D, Gofton C, Stephens S, Chaplin L, Christofidou-Solomidou M, Blake S et al. 1997. The in vivo and in vitro characterisation of an engineered human antibody to E-selectin. Immunotechnology, 3 (2), pp. 107-116. | Show Abstract | Read more

BACKGROUND: E-selectin is an endothelial cell specific adhesion molecule that is believed to play an important role in the early stages of leukocyte extravasation. OBJECTIVES: Here we describe the construction and evaluation of an engineered human monoclonal antibody that blocks E-selectin function. RESULTS: SPLAT-1 is an engineered human monoclonal antibody that has a very similar affinity for E-selectin as its murine parent antibody. In vitro SPLAT-1 blocks the binding of human leukocytes to E-selectin and does not mediate antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated lysis of endothelial cells. In vivo, SPLAT-1 inhibits the recruitment of leukocytes to cytokine-inflamed human skin grafted on to SCID mice and has a long circulating half-life in primates. It does not appear to provoke an immune response in primates even on repeat administration. CONCLUSIONS: SPLAT-1 has the characteristics of a antibody suitable for human therapy studies.

Ingram PE, Owens RJ, Woof JM. 1997. Development of a human IgA antibody specific for the tumour antigen TAG-72. Biochem Soc Trans, 25 (2), pp. 330S. | Read more

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Hughes B, Owens R, Perry M, Warrellow G, Allen R. 1997. PDE 4 inhibitors: The use of molecular cloning in the design and development of novel drugs DRUG DISCOVERY TODAY, 2 (3), pp. 89-101. | Show Abstract | Read more

Phosphodiesterase 4 (PDE 4) enzymes are the principal phosphodiesterases responsible for the hydrolysis of cAMP in pro-inflammatory leukocytes. The functional consequences of elevating cAMP in these cells suggests that inhibition of PDE 4 offers a novel approach to asthma therapy. However, clinical development of early inhibitors has been limited by the side-effect of nausea. In this review, we detail how the molecular biology of the PDE 4 gene family has been integrated with biochemical, cellular and pharmacological studies. This approach has led to the discovery and development of CDP840, a prototype inhibitor for which efficacy has been demonstrated in a clinical model of asthma in the absence of side-effects.

Shi J, Ghirlando R, Beavil RL, Beavil AJ, Keown MB, Young RJ, Owens RJ, Sutton BJ, Gould HJ. 1997. Interaction of the low-affinity receptor CD23/Fc epsilonRII lectin domain with the Fc epsilon3-4 fragment of human immunoglobulin E. Biochemistry, 36 (8), pp. 2112-2122. | Show Abstract | Read more

CD23/Fc epsilonRII, the low-affinity receptor for IgE, is a multifunctional protein of importance in blood cell development and the immune system. We have studied the interaction of CD23 with IgE in solution using hydrodynamic methods applied to recombinant fragments of both ligands: sCD23, corresponding to the soluble lectin domain of CD23, and Fc epsilon3-4, a dimer of the C epsilon3-C epsilon4 sequence of IgE. The hydrodynamic, spectroscopic, and biological properties of these fragments suggest that they have a fully native structure. Sedimentation equilibrium studies on mixtures of sCD23 and Fc epsilon3-4 indicate that IgE has two binding sites for CD23, each characterized by affinities of approximately 10(5) M(-1). Analysis of the sedimentation as a function of temperature allows conclusions to be drawn about the thermodynamics of binding at the two sites. Binding at the first site is characterized by large changes in enthalpy (delta H(degree)To = -2.1 +/- 3.3 kcal mol(-1)) and heat capacity (delta Cp(degree) = -320 +/- 320 cal mol(-1) K(-1)), whereas binding at the second site is characterized by small changes in enthalpy (delta H(degree)To = 0.1 +/- 5.6 kcal mol(-1)) and heat capacity (delta Cp(degree) = -140 +/- 550 cal mol(-1) K(-1)). In native CD23, there are two or three lectin domains, associated through an alpha-helical coiled-coil stalk. The predicted structure of the CD23 oligomers and symmetry considerations rule out the possibility of two lectin domains from one oligomer binding to identical sites in IgE. The notion of two types of interaction in the 2:1 complex between CD23 and IgE is consistent with the thermodynamic data presented.

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Cook JPD, Henry AJ, McDonnell JM, Owens RJ, Sutton BJ, Gould HJ. 1997. Identification of contact residues in the IgE binding site of human FcεRIα Biochemistry, 36 (50), pp. 15579-15588. | Show Abstract | Read more

The high-affinity receptor for immunoglobulin E (IgE), FcεRI, is an αβγ2 tetramer found on mast cells, basophils, and several other types of immune effector cells. The interaction of IgE with the α-subunit of FcεRI is central to the pathogenesis of allergy. Detailed knowledge of the mode of interaction of FcεRI with IgE may facilitate the development of inhibitors for general use in the treatment of allergic disease. To this end we have performed site-directed mutagenesis on a soluble form of the FcεRI α-chain (sFcεRIα). The effects of four mutations in the second immunoglobulin-like domain of sFcεRIα upon the kinetics of binding to IgE and fragments of IgE have been analyzed using surface plasmon resonance. As described in the preceding paper of this issue [Henry, A. J., et al. (1997) Biochemistry 36, 15568-15578], biphasic binding kinetics was observed. Two of the mutations had significant effects on binding: K117D reduced the affinity of sFcεRIα for IgE by a factor of 30, while D159K increased the affinity for IgE by a factor of 7, both principally through changes in the rates of dissociation of the slower phase of the interaction. Circular dichroism spectra of sFcεRIα incorporating either of these mutations were indistinguishable from those of wild-type sFcεRIα, demonstrating that the native conformation had not been disrupted. Our results, together with those from site-directed mutagenesis on fragments of IgE presented in the accompanying paper, define the contact surfaces in the IgE:sFcεRIα complex.

Ingram PE, Owens RJ, Woof JM. 1996. Development of a human IgA antibody specific for the tumor antigen TAG-72 IMMUNOLOGY, 89 pp. CC328-CC328.

Hughes B, Howat D, Lisle H, Holbrook M, James T, Gozzard N, Blease K, Hughes P, Kingaby R, Warrellow G et al. 1996. The inhibition of antigen-induced eosinophilia and bronchoconstriction by CDP840, a novel stereo-selective inhibitor of phosphodiesterase type 4. Br J Pharmacol, 118 (5), pp. 1183-1191. | Show Abstract | Read more

1. The novel tri-aryl ethane CDP840, is a potent and selective inhibitor of cyclic AMP phosphodiesterase type 4 (PDE 4) extracted from tissues or recombinant PDE 4 isoforms expressed in yeast (IC50S: 4-45 nM). CDP840 is stereo-selective since its S enantiomer (CT 1731) is 10-50 times less active against all forms of PDE 4 tested while both enantiomers are inactive (IC50S: > 100 microM) against PDE types 1, 2, 3 and 5. 2. Oral administration of CDP840 caused a dose-dependent reduction of interleukin-5 (IL-5)-induced pleural eosinophilia in rats (ED50 = 0.03 mg kg-1). The eosinophils in pleural exudates from CDP840-treated animals contained higher levels of eosinophil peroxidase (EPO) than cells from control animals, suggesting a stabilizing effect on eosinophil degranulation. CDP840 was approximately equi-active with the steroid dexamethasone in this model and was 10-100 times more potent than the known PDE 4-selective inhibitors rolipram and RP73401. The activity of CDP840 was not influenced by adrenalectomy, beta-sympathomimetics or beta-sympatholytics. 3. Antigen-induced pulmonary eosinophilia in sensitized guinea-pigs was reduced dose-dependently by CDP840 (0.01-1 mg kg-1, i.p.) and intracellular EPO levels were significantly higher. CDP840 was more potent in these activities than CT1731 or rolipram and comparable in potency to RP73401. 4. Rolipram or CDP840 were less active than dexamethasone in preventing neutrophil accumulation, or exudate formation in carrageenan-induced pleurisy in rats and thus do not exhibit general anti-inflammatory activity. 5. In sensitized guinea-pigs, aerosols of the antigen ovalbumin caused a dose-dependent bronchoconstriction demonstrated by an increase in pulmonary inflation pressure. Administration of CDP840 (0.001-1.0 mg kg-1, i.p.), 1 h before antigen challenge, resulted in dose-dependent reduction in response to antigen. This activity was not due to bronchodilatation since higher doses of CDP840 (3 mg kg-1) did not significantly change the bronchoconstrictor response to histamine. Rolipram was approximately 10 times less active than CDP840 in preventing antigen-induced bronchoconstriction. 6. These results confirm the observations that selective PDE 4 inhibitors reduce antigen-induced bronchoconstriction and pulmonary eosinophilic inflammation. CDP840 is more potent than rolipram in inhibiting native or recombinant PDE 4. Unlike the recently described potent PDE 4 inhibitor RP73401, CDP840 is more active than rolipram in the rat IL-5 model following oral administration. The novel series of tri-aryl ethanes, of which CDP840 is the lead compound, could be the basis of an orally active prophylactic treatment for human asthma.

Atkin JD, Pleass RJ, Owens RJ, Woof JM. 1996. Mutagenesis of the human IgA1 heavy chain tailpiece that prevents dimer assembly. J Immunol, 157 (1), pp. 156-159. | Show Abstract

The structural features of the human IgA1 tailpiece required for interaction with J chain in IgA dimer assembly were investigated using a protein engineering approach. Wild-type and mutant forms of IgA1 were expressed in the mouse myeloma cell line, J558L, which endogenously expresses J chain. Wild-type IgA1 was secreted as a mixture of dimers and monomers. Deletion of the entire tailpiece by stop codon introduction completely prevented dimer formation. Similarly, substitution of the penultimate residue of the tailpiece, Cys471, with serine resulted in the secretion of IgA monomers alone. Substitution of Asn459 with alanine to prevent attachment of N-linked carbohydrate to the tailpiece also resulted in markedly reduced dimer assembly. These results indicate the critical role played by the tailpiece, and Cys471 in particular, in IgA dimerization. In addition, we found tailpiece-deleted IgA1 and the Cys to Ser471 mutant IgA1 were secreted as mixtures of covalently associated monomers (alpha 2L2) and alpha L half-molecules. The tailpiece may thus play some role in promoting the association of alpha-chains required for IgA monomer assembly.

McCloskey N, Turner MW, Steffner P, Owens R, Goldblatt D. 1996. Human constant regions influence the antibody binding characteristics of mouse-human chimeric IgG subclasses. Immunology, 88 (2), pp. 169-173. | Show Abstract | Read more

Although antibody affinity is primarily determined by immunoglobulin variable region structure human IgG antibodies of the four subclasses specific for the same antigen have been shown to differ in their affinity. To explore the influence of the immunoglobulin constant region on functional antibody affinity, a set of V region identical mouse-human chimeric IgG subclasses specific for TAG72 (tumour-associated glycoprotein) were studied. Biomolecular interaction analysis (BIA) was used to determine the binding kinetics of whole IgG subclasses and F(ab')2 fragments. Despite identical V regions, binding kinetics differed for the four subclasses. The apparent dissociation rate constants of the intact immunoglobulins ranked IgG4 < IgG3 < IgG2 < IgG1. In contrast, analysis of the binding characteriztics of the F(ab')2 fragments derived from IgG1, IgG2 and IgG4 revealed identical binding kinetics. The structure of the constant regions of the humanized IgG subclass antibodies clearly influenced functional antibody affinity, as has been described for the murine IgG subclasses. The exact mechanism for this phenomenon remains obscure but such differences should be taken into account when designing or choosing antibodies for therapeutic use.

King DJ, Antoniw P, Owens RJ, Adair JR, Haines AM, Farnsworth AP, Finney H, Lawson AD, Lyons A, Baker TS. 1995. Preparation and preclinical evaluation of humanised A33 immunoconjugates for radioimmunotherapy. Br J Cancer, 72 (6), pp. 1364-1372. | Show Abstract | Read more

A humanised IgG1/k version of A33 (hA33) has been constructed and expressed with yields up to 700 mg l-1 in mouse myeloma NS0 cells in suspension culture. The equilibrium dissociation constant of hA33 (KD = 1.3 nM) was shown to be equivalent to that of the murine antibody in a cell-binding assay. hA33 labelled with yttrium-90 using the macrocyclic chelator 12N4 (DOTA) was shown to localise very effectively to human colon tumour xenografts in nude mice, with tumour levels increasing as blood concentration fell up to 144 h. A Fab' variant of hA33 with a single hinge thiol group to facilitate chemical cross-linking has also been constructed and expressed with yields of 500 mg l-1. Trimaleimide cross-linkers have been used to produce a trivalent Fab fragment (hA33 TFM) that binds antigen on tumour cells with greater avidity than hA33 IgG. Cross-linkers incorporating 12N4 or 9N3 macrocycles have been used to produce hA33 TFM labelled stably and site specifically with yttrium-90 or indium-111 respectively. These molecules have been used to demonstrate that hA33 TFM is cleared more rapidly than hA33 IgG from the circulation of animals but does not lead to accumulation of these metallic radionuclides in the kidney. 90Y-labelled hA33 TFM therefore appears to be the optimal form of the antibody for radioimmunotherapy of colorectal carcinoma.

HUGHES B, HOLBROOK M, ALLEN R, OWENS R, PERRY M, WARRELLOW G, HOWAT D, BLOXHAM D, HIGGS G. 1995. SUPPRESSION OF ANTIGEN-INDUCED AIRWAY RESPONSES AND BRONCHIAL HYPERRESPONSIVENESS BY CDP840, A NOVEL STEREOSELECTIVE INHIBITOR OF PHOSPHODIESTERASE TYPE-IV BRITISH JOURNAL OF PHARMACOLOGY, 116 pp. P6-P6.

Keown MB, Ghirlando R, Young RJ, Beavil AJ, Owens RJ, Perkins SJ, Sutton BJ, Gould HJ. 1995. Hydrodynamic studies of a complex between the Fc fragment of human IgE and a soluble fragment of the Fc epsilon RI alpha chain. Proc Natl Acad Sci U S A, 92 (6), pp. 1841-1845. | Show Abstract | Read more

The interaction between immunoglobulin E (IgE) and its high-affinity receptor Fc epsilon RI is central to allergic disease. The binding site for Fc epsilon RI lies in the third constant region domain of the epsilon heavy chain of IgE (C epsilon 3). Identical epitopes on the two C epsilon 3 domains in the IgE-Fc are predicted to be on opposite sides of the structure, and therefore each could bind independently to a receptor molecule. Titrations, however, reveal that the IgE-Fc forms an equimolar complex with a soluble fragment of the Fc epsilon RI alpha chain (sFc epsilon RI alpha), and the molecular weight of the complex, as determined by sedimentation equilibrium, confirms this stoichiometry. The measured sedimentation coefficients of the two ligands are in good agreement with computed values for a compact IgE-Fc and an elongated sFc epsilon RI alpha structure. The calculated sedimentation coefficients for possible models of a 1:1 complex lead to exclusion of all highly extended geometries for the complex. Possible explanations for the paradoxical stoichiometry of the IgE-Fc/sFc epsilon RI alpha complex, in terms of the curved shape of IgE, a conformational change in IgE when the receptor binds, and steric interference between two molecules of Fc epsilon RI binding to identical sites, are discussed.

Young RJ, Owens RJ, Mackay GA, Chan CM, Shi J, Hide M, Francis DM, Henry AJ, Sutton BJ, Gould HJ. 1995. Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants. Protein Eng, 8 (2), pp. 193-199. | Show Abstract | Read more

We have constructed an expression vector that leads to secretion of the whole Fc of human immunoglobulin E (hIgE-Fc) from mammalian cells at levels up to 100 mg/l of culture. Two surface glycosylation sites at Asn265 and Asn371 have been changed to glutamine, to obtain a more homogeneous preparation of hIgE-Fc for structural studies. Comparison of wild-type and mutant products revealed that Asn371 is rarely glycosylated in Chinese hamster ovary cells. Both the double mutant and wild-type hIgE-Fc bind to the high-affinity IgE receptor, Fc epsilon RI, with about the same affinity as myeloma IgE (Ka in the range 10(10)-10(11) M-1), and were able to sensitize isolated human basophils for anti-IgE triggering of histamine release. However, only the double mutant hIgE-Fc approached the affinity of myeloma IgE for the low-affinity receptor, Fc epsilon RII (Ka = 7.3 x 10(7) M-1), whereas the wild-type hIgE-Fc bound with a 10-fold lower affinity (Ka = 4.1 x 10(6) M-1).

King DJ, Turner A, Farnsworth AP, Adair JR, Owens RJ, Pedley RB, Baldock D, Proudfoot KA, Lawson AD, Beeley NR. 1994. Improved tumor targeting with chemically cross-linked recombinant antibody fragments. Cancer Res, 54 (23), pp. 6176-6185. | Show Abstract

The construction and use of recombinant chimeric and later fully humanized (CDR-grafted) antibodies to tumor-associated antigens has reduced the immune response generated to these antibodies in clinical studies. However, their long circulating half-life is a disadvantage for tumor imaging and therapy. Fragments such as F(ab')2, Fab', Fv and single chain Fv (scFv) offer faster blood clearance but also lower overall tumor doses. We have examined the tumor targeting of several novel fragments produced by chemical cross-linking of Fab' or scFv to dimeric and trimeric species. To facilitate cross-linking of Fab' fragments, a chimeric B72.3 Fab' fragment has been expressed with a hinge sequence containing a single cysteine residue. B72.3 scFv was also produced with a similar hinge region peptide attached to the COOH terminus to allow cross-linking. These fragments, Fab' delta Cys and scFv' delta Cys were cross-linked with linkers containing two or three maleimide groups to produce dimeric and trimeric molecules with increased avidity for antigen. Cross-linkers were also designed to contain a 12-N-4 macrocycle capable of stable radiolabeling with 90Y. This allowed the production of site-specifically-labeled, fully immunoreactive proteins. Biodistribution studies in the nude mouse LS174T xenograft model with scFv, di-scFv, and tri-scFv demonstrated that these fragments clear extremely rapidly from the circulation and give rise to only low levels of activity accumulated at the tumor. Di-Fab (DFM) and tri-Fab (TFM) however, accumulated relatively high levels of activity at the tumor with high tumor:blood ratios generated, demonstrating improved targeting compared to IgG. cB72.3 90Y-labeled tri-Fab was found not to accumulate in the kidney or the bone, resulting in an attractive antibody fragment for tumor therapy.

Morelock MM, Rothlein R, Bright SM, Robinson MK, Graham ET, Sabo JP, Owens R, King DJ, Norris SH, Scher DS. 1994. Isotype choice for chimeric antibodies affects binding properties. J Biol Chem, 269 (17), pp. 13048-13055. | Show Abstract

Construction of a series of chimeric antibodies (murine variable region and human constant region) derived from the murine antibody BIRR1, which recognizes intercellular adhesion molecule 1 (ICAM-1), has revealed differences in the relative binding abilities of the chimeric antibody to antigen. The chimeric antibodies show a ranking of their ability to compete with BIRR1 for antigen on the surface of cells with the order BIRR1 = cIgG1 (100%) > cIgG4 (30%) > cIgG2 (10%) as demonstrated by solid-phase competitive enzyme-linked immunosorbent assay. Papain digestion yielded Fab fragments that were purified to homogeneity. Competitive enzyme-linked immunosorbent assay showed that the chimeric and murine Fab binding constants were equivalent. A solution-phase binding assay (analyzed by size exclusion high performance liquid chromatography) between the intact mAbs and recombinant soluble ICAM-1 further established that the binding constants involving the Fab arms of the two antibodies were equivalent. In summary, the murine and chimeric anti-ICAM-1 antibodies bind cellular ICAM-1 with equivalent affinities but with differing avidities.

Owens RJ, Young RJ. 1994. The genetic engineering of monoclonal antibodies. J Immunol Methods, 168 (2), pp. 149-165. | Show Abstract | Read more

A number of recent technological developments have greatly facilitated the genetic engineering of immunoglobulins. The use of PCR has permitted the variable regions to be rapidly cloned either from a specific hybridoma source or as a gene library from non-immunised cells. The conversion of the rodent antibody into a humanized version is now well established. To develop these antibodies for clinical use has required the development of high level expression systems. For the expression of large multimeric glycoproteins, mammalian cell systems generally provide the highest levels of secreted product and therefore are the methods of choice for producing whole recombinant antibodies. Novel antigen-binding units have been developed by joining the two variable domains of an antibody into single-chain polypeptides. Such fragments can be produced in high yield by secretion from E. coli raising the prospect of bulk preparation of these antibody fragments for the development of low-cost immunopurification and assay reagents. Finally, the ability to screen for antigen binding by displaying immunoglobulin variable regions on the surface of filamentous bacteriaphages has opened up the possibility of bypassing the immune system to generate novel antibody specificities in vitro.

Baker TS, Bose CC, Caskey-Finney HM, King DJ, Lawson AD, Lyons A, Mountain A, Owens RJ, Rolfe MR, Sehdev M. 1994. Humanization of an anti-mucin antibody for breast and ovarian cancer therapy. Adv Exp Med Biol, 353 pp. 61-82. | Show Abstract | Read more

Antibody-drug conjugates utilize the targetting potential of antibodies to improve the potential of cytostatic or cytocidal drugs. One such murine monoclonal antibody, CTM01 (mCTM01), which recognizes an epitope on breast epithelial mucin, has potential for the treatment of breast and ovarian cancers. We examine in this paper the comparative properties of mCTM01 against a number of other anti-mucin antibodies. We then describe the humanization and high level re-expression of humanized CTM01 (hCTM01), a process designed to avoid the immune response to administered murine antibodies in human patients and to produce sufficient material for clinical studies. We show that the humanized form has properties superior to mCTM01 in terms of binding affinity to antigen presented on tumour cells.

Beavil AJ, Beavil RL, Chan CM, Cook JP, Gould HJ, Henry AJ, Owens RJ, Shi J, Sutton BJ, Young RJ. 1993. Structural basis of the IgE-Fc epsilon RI interaction. Biochem Soc Trans, 21 (4), pp. 968-972. | Read more

Morton HC, Atkin JD, Owens RJ, Woof JM. 1993. Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells. J Immunol, 151 (9), pp. 4743-4752. | Show Abstract

Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fc alpha receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.

King DJ, Byron OD, Mountain A, Weir N, Harvey A, Lawson AD, Proudfoot KA, Baldock D, Harding SE, Yarranton GT. 1993. Expression, purification and characterization of B72.3 Fv fragments. Biochem J, 290 ( Pt 3) (3), pp. 723-729. | Show Abstract | Read more

The Fv fragment of the antibody B72.3 has been produced by expression in both a mammalian and microbial system, namely Chinese hamster ovary (CHO) cells and Escherichia coli. In both cases secretion of the Fv into the culture medium was achieved, with equivalent amounts of Vh and Vl produced. The yield of Fv from CHO cells was 4 mg/l in roller-bottle culture. E. coli proved to be a more productive system with yields of 40 mg/l in shake flasks rising to 450 mg/l in fermentations. B72.3 Fv from both sources was capable of binding to antigen with similar binding ability to the Fab' fragment. A detailed sedimentation analysis, both by velocity and equilibrium techniques, revealed that the two domains of Fv are associated at high concentrations at pH values close to neutral, but dissociate at concentrations lower than approx. 0.5 mg/ml. Individual Vh or Vl polypeptides are not able to bind to the antigen and thus these results suggest that the antigen promotes assembly of Fv at the low concentrations used in the antigen-binding assays. At a pH value of 1.9, Vh and Vl are completely dissociated even at very high concentrations and are apparently unfolded at low solute concentrations. Small-angle X-ray scattering was used to measure a radius of gyration of 1.75 +/- 0.2 nm (17.5 +/- 2 A) for Fv.

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KING DJ, MOUNTAIN A, ADAIR JR, OWENS RJ, HARVEY A, WEIR N, PROUDFOOT KA, PHIPPS A, LAWSON A, RHIND SK et al. 1992. TUMOR-LOCALIZATION OF ENGINEERED ANTIBODY FRAGMENTS ANTIBODY IMMUNOCONJUGATES AND RADIOPHARMACEUTICALS, 5 (2), pp. 159-170.

Lyons A, King DJ, Owens RJ, Yarranton GT, Millican A, Whittle NR, Adair JR. 1990. Site-specific attachment to recombinant antibodies via introduced surface cysteine residues. Protein Eng, 3 (8), pp. 703-708. | Show Abstract | Read more

Many diagnostic and therapeutic applications of monoclonal antibodies require the covalent linking of effector or reporter molecules to the immunoglobulin polypeptides. Existing methods generally involve the non-selective modification of amino acid side chains, producing one or more randomly distributed attachment sites. This results in heterogeneous labelling of the antibody molecules and often to a decrease in antigen-binding due to the modification of residues close to the antigen-binding site. We report a novel strategy for site-specifically labelling antibodies through surface cysteine residues. Examination of molecular structures was used to identify amino acids of the CH1 domain of the IgG heavy chain that were accessible to solvent but not to larger molecules. Site-directed mutagenesis was used to substitute cysteine residues at these positions in the heavy chain of a mouse/human chimaeric version of the tumour-binding monoclonal antibody, B72.3. Expression of the modified antibody genes in mammalian cells yielded correctly assembled proteins that had thiol groups in pre-determined positions and showed no loss of antigen-binding activity. One of the mutants was used to demonstrate the site-specific attachment of a radio-iodinated ligand to the chimaeric B72.3 antibody.

Crompton MR, Owens RJ, Totty NF, Moss SE, Waterfield MD, Crumpton MJ. 1988. Primary structure of the human, membrane-associated Ca2+-binding protein p68 a novel member of a protein family. EMBO J, 7 (1), pp. 21-27. | Show Abstract | Read more

cDNA clones encoding human 'p68', a membrane-associated Ca2+-binding protein, were isolated from a lambda gt11 expression library of the human T-leukaemia cell line J6, by using a rabbit antiserum against denatured purified lymphocyte p68, and from a liver cDNA library by using 32P-labelled p68 cDNA fragments. The amino acid sequence of p68, deduced from the sequences of overlapping cDNA clones, is described. The results show that p68 is closely related to a family of proteins which includes intracellular substrates of the EGF receptor and pp60src tyrosine kinases. The p68 amino acid sequence is internally repetitive, being constructed from eight repeats of varying lengths, each of which contains a highly conserved sequence. Multiple copies of the latter sequence are also present in the related proteins p36, lipocortin I and protein II. We discuss how the common structural features of these proteins may reflect common functions and, furthermore, how the eight repeat structure of p68 may have evolved. The sequences of independent cDNAs suggest that alternatively-spliced mRNAs could encode different p68 protein species. This suggestion is consistent with the observation that purified p68 migrates as a closely-spaced doublet when analysed by SDS-PAGE.

Carnemolla B, Borsi L, Zardi L, Owens RJ, Baralle FE. 1987. Localization of the cellular-fibronectin-specific epitope recognized by the monoclonal antibody IST-9 using fusion proteins expressed in E. coli. FEBS Lett, 215 (2), pp. 269-273. | Show Abstract | Read more

Here we report on a monoclonal antibody (IST-9) which distinguishes between human cellular and plasma fibronectin. Using beta-galactosidase-fibronectin fusion proteins expressed in E. coli we have demonstrated that this monoclonal antibody is specific for a fibronectin segment (ED) which can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Furthermore, using the same fusion proteins we have been able to localize precisely the epitopes of two other monoclonal antibodies (IST-1 and IST-2), specific for the heparin-binding domain 5 of fibronectin.

Owens RJ, Kornblihtt AR, Baralle FE. 1986. Fibronectin, the generation of multiple polypeptides from a single gene. Oxf Surv Eukaryot Genes, 3 pp. 141-160.

Owens RJ, Baralle FE. 1986. Mapping the collagen-binding site of human fibronectin by expression in Escherichia coli. EMBO J, 5 (11), pp. 2825-2830. | Show Abstract | Read more

The collagen-binding domain of human fibronectin has been expressed as a cro/beta-galactosidase fusion protein in Escherichia coli. The hybrid polypeptide was recognized by an anti-(human plasma fibronectin) serum and bound specifically to gelatin-Sepharose. The collagen-binding region was subdivided by constructing a series of overlapping bacterial expression plasmids. The fusion proteins produced by these constructs were analysed for gelatin-binding activity. The results indicate that the binding site lies within an approximately 12.5 kd fragment of fibronectin, and show that the following 14 amino acid sequence is critical for gelatin-binding activity: Ala-Ala-His-Glu-Glu-Ile-Cys-Thr-Thr-Asn-Glu-Gly-Val-Met. This sequence links the second type II homology unit with the adjacent type I repeat in the amino-terminal third of the fibronectin molecule.

Owens RJ, Baralle FE. 1986. Exon structure of the collagen-binding domain of human fibronectin. FEBS Lett, 204 (2), pp. 318-322. | Show Abstract | Read more

Fibronectin consists of a series of three internal homology repeats (types I, II, and III [(1983) Proc. Natl. Acad. Sci. USA 80, 137-141]). The type III units are each coded for by two exons with the exception of the alternatively spliced extra domain type III [(1984) EMBO J. 3, 2511-2516]. To investigate the gene organisation of the type I and II repeats, genomic clones covering the collagen-binding domain of human fibronectin have been isolated and characterised. The results show that each of the type I and II homology units in this region corresponds to an exon. Taken together with the previous data concerning the type III units, the data lend support to a gene fusion model of fibronectin evolution.

OWENS RJ, CRUMPTON MJ. 1984. A NEW CLASS OF MEMBRANE-ASSOCIATED CALCIUM-BINDING PROTEINS BIOESSAYS, 1 (2), pp. 61-63. | Show Abstract | Read more

Calcium ions act as modulators of many fundamental processes in eukaryotic cells. Although these processes apparently involve initial interactions between calcium ions and cell membranes, the identity of the putative membrane Ca2+‐binding proteins has until recently been obscure. This article describes a recently discovered family of mammalian membrane proteins, of perhaps ancient origin, that may fulfil this function. Copyright © 1984 ICSU Press

Owens RJ, Gallagher CJ, Crumpton MJ. 1984. Cellular distribution of p68, a new calcium-binding protein from lymphocytes. EMBO J, 3 (5), pp. 945-952. | Show Abstract | Read more

A Ca2+-binding protein of mol. wt. 68 000 ( p68 ) is a major component of a Nonidet P-40 insoluble fraction of human and pig lymphocyte plasma membrane. An affinity-purified rabbit antibody has been produced against p68 and used to study its cellular distribution. The antibody stained fixed and permeabilised human B lymphoblastoid cells, peripheral blood lymphocytes and sections of human tonsil. Whole cells, however, were not stained, indicating that the protein was not represented at the cell surface. This assignment was consistent with the detection of p68 in immunoprecipitates from biosynthetically- but not surface-labelled cells. It is concluded that p68 is located on the cytoplasmic face of the plasma membrane. Subcellular fractionation experiments confirmed that p68 was largely membrane-bound in lymphocytes, although a small soluble fraction (approximately 10% of the total) was detected. Sub-fractionation of lymphocyte membranes revealed that p68 was associated not only with the plasma membrane but also with other endomembrane systems. As judged by immunoprecipitation, p68 was present in a variety of cultured cell lines of both lymphoid and non-lymphoid origin. p68 demonstrated a diffuse distribution in fixed and permeabilised fibroblasts which did not correspond to the distribution of either microfilaments or intermediate filaments. However, in detergent-extracted cells the protein was localised in a lamina-like network. A similar immunofluorescent staining pattern has recently been observed for spectrin-related proteins in the detergent-resistant cytoskeleton of fibroblasts. It is suggested that p68 is part of a sub-membranous cytoskeletal complex not only in lymphocytes but also in other cell types.

GALLAGHER CJ, OWENS RJ, CRUMPTON MJ. 1984. INTRACELLULAR MARKERS OF DIFFERENTIATION IN BURKITTS-LYMPHOMA PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, 25 (MAR), pp. 46-46.

Davies AA, Wigglesworth NM, Allan D, Owens RJ, Crumpton MJ. 1984. Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue. Biochem J, 219 (1), pp. 301-308. | Show Abstract | Read more

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5'-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

Owens RJ, Crumpton MJ. 1984. Isolation and characterization of a novel 68,000-Mr Ca2+-binding protein of lymphocyte plasma membrane. Biochem J, 219 (1), pp. 309-316. | Show Abstract | Read more

A 68 000-Mr protein is a major component of a Nonidet P-40-insoluble fraction of lymphocyte plasma membrane prepared from human B lymphoblastoid cells ( BRI 8) and pig mesenteric lymph nodes. The association of the protein with the detergent-insoluble complex depends on free Ca2+ concentrations of greater than 10 microM. The human and pig 68 000-Mr proteins were purified and appear to be homologous on the basis of amino acid composition and peptide mapping. The protein is monomeric, has pI 5.8 and a single high-affinity Ca2+-binding site (KD 1.2 microM). The results are discussed in terms of the possible role of the 68 000-Mr protein as an intracellular Ca2+ receptor in lymphocytes.

Crumpton MJ, Owens RJ, Gallagher CJ, Davies AA. 1983. The cell surface and its metabolism. J Pathol, 141 (3), pp. 235-248. | Show Abstract | Read more

The cell surface structure is highly dynamic. In particular, binding of ligand induces the redistribution of receptors on the cell surface as well as the internalisation of ligand-receptor complexes. Internalisation in turn leads to a recycling of the receptor or to a decrease in the cell's responsiveness to the ligand. Modulation of the cell surface structure is apparently regulated intracellularly by components of the cell's cytoskeleton. A crucial component in this respect is likely to be a sub-membranous filamentous network that is linked directly to the cytoplasmic face of the surface membrane. In erythrocytes this network can be separated from purified preparations of the plasma membrane by virtue of its insolubility in nonionic detergents. Application of this procedure to the plasma membrane fraction of human B lymphoblastoid cells has yielded a detergent-insoluble residue comprising actin and a 68,000-Mr polypeptide as major components, together with polypeptides of 28,000-, 33,000- and 120,000-Mr as prominent but more minor components. The association of the 68,000-Mr protein with the detergent-insoluble residue and the original plasma membrane is Ca2+-dependent. Burkitt lymphoma cells differ noticeably from lymphoblastoid cells in that the 68,000-Mr protein is not associated with the inner face of the surface membrane. This difference may reflect the malignant phenotype of Burkitt lymphomas or the hypothetical sub-population of normal B lymphocytes from which the lymphomas are derived.

OWENS RAYMONDJ, CRUMPTON MICHAELJ. 1983. A new cytoskeletal protein associated with the plasma membrane of lymphocytes Biochemical Society Transactions, 11 (2), pp. 156-157. | Read more

Owens RJ, Northcote DH. 1981. The location of arabinosyl:hydroxyproline transferase in the membrane system of potato tissue culture cells. Biochem J, 195 (3), pp. 661-667. | Show Abstract | Read more

Incubation of a particulate preparation from potato tissue culture cells with UDP-beta-L-[1-3H] arabinose yielded a glycoprotein fraction containing labelled material with the characteristics of hydroxyproline arabinosides. The sugar-protein linkage was resistant to hot alkaline hydrolysis, and the hydrolytic products showed similar electrophoretic and chromatographic behavior to authentic hydroxyproline-arabinosides prepared from potato tissue culture cell walls. Incorporation of arabinose into glycoprotein was stimulated by the addition of de-arabinosylated potato lectin. The product of the incubation co-migrated with native potato lectin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The subcellular distribution of the arabinosyl-transferase was investigated by fractionating potato tissue culture membranes on a discontinuous sucrose gradient in the presence or absence of Mg2+. Under both fractionation conditions the highest specific activity of the enzyme was found in the Golgi-enriched fraction. The results are discussed in relation to the synthesis of the hydroxy-proline-rich glycoprotein component of plant cell walls.

JUNIPER BE, OWENS RJ, WHALE D, SHAW AC. 1980. THE REDISTRIBUTION OF REDUCING POWER IN THE ELONGATING ZONE OF GEOSTIMULATED BEAN ROOTS PLANT SCIENCE LETTERS, 18 (2), pp. 127-132. | Show Abstract | Read more

Beans (Vicia faba) were grown in darkness until their roots were 20-40 mm in length. Still in the dark they were then placed horizontally and after specific times the terminal 20 mm of the roots was excised, bisected in a plane at right angles to the direction of gravity and the (new) upper and lower half segments were analysed for reducing power by the dichlorophenolindophenol method. At approximately three hours after having been placed in the horizontal position there was a marked decrease of approx. 20% of the reducing power in the upper half segment of the root followed by an equally rapid return to equivalence. The time of this loss corresponds closely with the maximum period of the bending. If the horizontal roots were not bisected and then compared to bisected tips there was no significant difference in total reducing power. This suggests that the loss of reducing power in the upper half was not due to 'leakage' as a result of the surgery. © 1980.

OWENS RJ, NORTHCOTE DH. 1980. THE PURIFICATION OF POTATO LECTIN BY AFFINITY-CHROMATOGRAPHY ON A FETUIN-SEPHAROSE MATRIX PHYTOCHEMISTRY, 19 (8), pp. 1861-1862. | Read more

Ren J, Nettleship JE, Males A, Stuart DI, Owens RJ. 2016. Crystal structures of penicillin-binding protein 3 in complexes with azlocillin and cefoperazone in both acylated and deacylated forms. FEBS Lett, 590 (2), pp. 288-297. | Show Abstract | Read more

Penicillin-binding protein 3 (PBP3) from Pseudomonas aeruginosa is the molecular target of β-lactam-based antibiotics. Structures of PBP3 in complexes with azlocillin and cefoperazone, which are in clinical use for the treatment of pseudomonad infections, have been determined to 2.0 Å resolution. Together with data from other complexes, these structures identify a common set of residues involved in the binding of β-lactams to PBP3. Comparison of wild-type and an active site mutant (S294A) showed that increased thermal stability of PBP3 following azlocillin binding was entirely due to covalent binding to S294, whereas cefoperazone binding produces some increase in stability without the covalent link. Consistent with this, a third crystal structure was determined in which the hydrolysis product of cefoperazone was noncovalently bound in the active site of PBP3. This is the first structure of a complex between a penicillin-binding protein and cephalosporic acid and may be important in the design of new noncovalent PBP3 inhibitors.

Arena de Souza V, Scott DJ, Nettleship JE, Rahman N, Charlton MH, Walsh MA, Owens RJ. 2015. Comparison of the Structure and Activity of Glycosylated and Aglycosylated Human Carboxylesterase 1. PLoS One, 10 (12), pp. e0143919. | Show Abstract | Read more

Human Carboxylesterase 1 (hCES1) is the key liver microsomal enzyme responsible for detoxification and metabolism of a variety of clinical drugs. To analyse the role of the single N-linked glycan on the structure and activity of the enzyme, authentically glycosylated and aglycosylated hCES1, generated by mutating asparagine 79 to glutamine, were produced in human embryonic kidney cells. Purified enzymes were shown to be predominantly trimeric in solution by analytical ultracentrifugation. The purified aglycosylated enzyme was found to be more active than glycosylated hCES1 and analysis of enzyme kinetics revealed that both enzymes exhibit positive cooperativity. Crystal structures of hCES1 a catalytically inactive mutant (S221A) and the aglycosylated enzyme were determined in the absence of any ligand or substrate to high resolutions (1.86 Å, 1.48 Å and 2.01 Å, respectively). Superposition of all three structures showed only minor conformational differences with a root mean square deviations of around 0.5 Å over all Cα positions. Comparison of the active sites of these un-liganded enzymes with the structures of hCES1-ligand complexes showed that side-chains of the catalytic triad were pre-disposed for substrate binding. Overall the results indicate that preventing N-glycosylation of hCES1 does not significantly affect the structure or activity of the enzyme.

Tiede C, Tang AAS, Deacon SE, Mandal U, Nettleship JE, Owen RL, George SE, Harrison DJ, Owens RJ, Tomlinson DC, McPherson MJ. 2014. Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications. Protein Eng Des Sel, 27 (5), pp. 145-155. | Show Abstract | Read more

We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 10(10) clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications.

van Berkel SS, Nettleship JE, Leung IKH, Brem J, Choi H, Stuart DI, Claridge TDW, McDonough MA, Owens RJ, Ren J, Schofield CJ. 2013. Binding of (5S)-penicilloic acid to penicillin binding protein 3. ACS Chem Biol, 8 (10), pp. 2112-2116. | Show Abstract | Read more

β-Lactam antibiotics react with penicillin binding proteins (PBPs) to form relatively stable acyl-enzyme complexes. We describe structures derived from the reaction of piperacillin with PBP3 (Pseudomonas aeruginosa) including not only the anticipated acyl-enzyme complex but also an unprecedented complex with (5S)-penicilloic acid, which was formed by C-5 epimerization of the nascent (5R)-penicilloic acid product. Formation of the complex was confirmed by solution studies, including NMR. Together, these results will be useful in the design of new PBP inhibitors and raise the possibility that noncovalent PBP inhibition by penicilloic acids may be of clinical relevance.

Nettleship JE, Ren J, Scott DJ, Rahman N, Hatherley D, Zhao Y, Stuart DI, Barclay AN, Owens RJ. 2013. Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment. BMC Struct Biol, 13 (1), pp. 13. | Show Abstract | Read more

BACKGROUND: Signal Regulatory Protein γ (SIRPγ) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRPγ is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRPγ. RESULTS: We obtained crystals of SIRPγ by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRPα and SIRPβ. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRPγ and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRPγ and SIRPα but not SIRPβ. Comparison of the three domain structures of SIRPγ and SIRPα showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRPγ in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRPγ were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRPγ: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd = 1.2 +/- 0.3 μM). CONCLUSION: The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRPγ stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution.

Nettleship JE, Flanagan A, Rahman-Huq N, Hamer R, Owens RJ. 2012. Converting monoclonal antibodies into Fab fragments for transient expression in mammalian cells. Methods Mol Biol, 801 pp. 137-159. | Show Abstract | Read more

In this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5' primers that hybridise to the first framework sequence of the mouse light and heavy chains and 3' primers that bind to the heavy- and light-chain constant regions. The amplified sequences are inserted into mammalian cell expression vectors by In-Fusion™ cloning. This method allows vector and amplified DNA sequences to be seamlessly joined in a ligation-independent reaction. Transient co-expression of light-chain and heavy-chain genes in HEK 293T cells enables production of recombinant Fabs for functional and structural studies.

Green VL, Verma A, Owens RJ, Phillips SEV, Carr SB. 2011. Structure of New Delhi metallo-β-lactamase 1 (NDM-1). Acta Crystallogr Sect F Struct Biol Cryst Commun, 67 (Pt 10), pp. 1160-1164. | Show Abstract | Read more

Antibiotic resistance in bacterial pathogens poses a serious threat to human health and the metallo-β-lactamase (MBL) enzymes are responsible for much of this resistance. The recently identified New Delhi MBL 1 (NDM-1) is a novel member of this family that is capable of hydrolysing a wide variety of clinically important antibiotics. Here, the crystal structure of NDM-1 from Klebsiella pneumoniae is reported and its structure and active site are discussed in the context of other recently deposited coordinates of NDM-1.

Holdom MD, Davies AM, Nettleship JE, Bagby SC, Dhaliwal B, Girardi E, Hunt J, Gould HJ, Beavil AJ, McDonnell JM et al. 2011. Conformational changes in IgE contribute to its uniquely slow dissociation rate from receptor FcɛRI. Nat Struct Mol Biol, 18 (5), pp. 571-576. | Show Abstract | Read more

Among antibody classes, IgE has a uniquely slow dissociation rate from, and high affinity for, its cell surface receptor FcɛRI. We show the structural basis for these key determinants of the ability of IgE to mediate allergic hypersensitivity through the 3.4-Å-resolution crystal structure of human IgE-Fc (consisting of the Cɛ2, Cɛ3 and Cɛ4 domains) bound to the extracellular domains of the FcɛRI α chain. Comparison with the structure of free IgE-Fc (reported here at a resolution of 1.9 Å) shows that the antibody, which has a compact, bent structure before receptor engagement, becomes even more acutely bent in the complex. Thermodynamic analysis indicates that the interaction is entropically driven, which explains how the noncontacting Cɛ2 domains, in place of the flexible hinge region of IgG antibodies, contribute together with the conformational changes to the unique binding properties of IgE.

Sainsbury S, Bird L, Rao V, Shepherd SM, Stuart DI, Hunter WN, Owens RJ, Ren J. 2011. Crystal structures of penicillin-binding protein 3 from Pseudomonas aeruginosa: comparison of native and antibiotic-bound forms. J Mol Biol, 405 (1), pp. 173-184. | Show Abstract | Read more

We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft. The orientations of the two β-lactams in the active site and the key interactions formed between the ligands and PBP3 are similar despite differences in the two drugs, indicating a degree of flexibility in the binding site. The conserved binding mode of β-lactam-based inhibitors appears to extend to other PBPs, as suggested by a comparison of the PBP3/ceftazidime complex and the Escherichia coli PBP1b/ceftoxamine complex. Since P. aeruginosa is an important human pathogen, the structural data reveal the mode of action of the frontline antibiotic ceftazidime at the molecular level. Improved drugs to combat infections by P. aeruginosa and related Gram-negative bacteria are sought and our study provides templates to assist that process and allows us to discuss new ways of inhibiting PBPs.

Nettleship JE, Assenberg R, Diprose JM, Rahman-Huq N, Owens RJ. 2010. Recent advances in the production of proteins in insect and mammalian cells for structural biology. J Struct Biol, 172 (1), pp. 55-65. | Show Abstract | Read more

The production of proteins in sufficient quantity and of appropriate quality is an essential pre-requisite for structural studies. Escherichia coli remains the dominant expression system in structural biology with nearly 90% of the structures in the Protein Data Bank (PDB) derived from proteins produced in this bacterial host. However, many mammalian and eukaryotic viral proteins require post-translation modification for proper folding and/or are part of large multimeric complexes. Therefore expression in higher eukaryotic cell lines from both invertebrate and vertebrate is required to produce these proteins. Although these systems are generally more time-consuming and expensive to use than bacteria, there have been improvements in technology that have streamlined the processes involved. For example, the use of multi-host vectors, i.e., containing promoters for not only E. coli but also mammalian and baculovirus expression in insect cells, enables target genes to be evaluated in both bacterial and higher eukaryotic hosts from a single vector. Culturing cells in micro-plate format allows screening of large numbers of vectors in parallel and is amenable to automation. The development of large-scale transient expression in mammalian cells offers a way of rapidly producing proteins with relatively high throughput. Strategies for selenomethionine-labelling (important for obtaining phase information in crystallography) and controlling glycosylation (important for reducing the chemical heterogeneity of glycoproteins) have also been reported for higher eukaryotic cell expression systems.

Sainsbury S, Lane LA, Ren J, Gilbert RJ, Saunders NJ, Robinson CV, Stuart DI, Owens RJ. 2009. The structure of CrgA from Neisseria meningitidis reveals a new octameric assembly state for LysR transcriptional regulators. Nucleic Acids Res, 37 (14), pp. 4545-4558. | Show Abstract | Read more

LysR-type transcriptional regulators (LTTRs) form the largest family of bacterial regulators acting as both auto-repressors and activators of target promoters, controlling operons involved in a wide variety of cellular processes. The LTTR, CrgA, from the human pathogen Neisseria meningitidis, is upregulated during bacterial-host cell contact. Here, we report the crystal structures of both regulatory domain and full-length CrgA, the first of a novel subclass of LTTRs that form octameric rings. Non-denaturing mass spectrometry analysis and analytical ultracentrifugation established that the octameric form of CrgA is the predominant species in solution in both the presence and absence of an oligonucleotide encompassing the CrgA-binding sequence. Furthermore, analysis of the isolated CrgA-DNA complex by mass spectrometry showed stabilization of a double octamer species upon DNA binding. Based on the observed structure and the mass spectrometry findings, a model is proposed in which a hexadecameric array of two CrgA oligomers binds to its DNA target site.

Nettleship JE, Rahman-Huq N, Owens RJ. 2009. The production of glycoproteins by transient expression in Mammalian cells. Methods Mol Biol, 498 pp. 245-263. | Show Abstract | Read more

In this chapter, protocols for the growth and transfection of Human Embryonic Kidney (HEK) 293T cells for small scale expression screening and large scale protein production are described. Transient expression in mammalian cells offers a method of rapidly producing glycoproteins with a relatively high throughput. HEK 293T cells, in particular, can be transfected with high efficiency (> 50% cell expression) and are amenable to culture at multi-litre scale. Growing cells in micro-plate format allows screening of large numbers of vectors in parallel to prioritise those amenable to scale-up and purification for subsequent structural or functional studies. The glycoform of the expressed protein can be modified by treating cell cultures with kifunensine which inhibits glycan processing during protein synthesis. This results in the production of a chemically homogeneous glycoprotein with short mannose-rich sugar chains attached to the protein backbone. If required, these can be readily removed by endoglycosidase treatment.

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