Dr Maria Harkiolaki
| Research Area: | Protein Science and Structural Biology |
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| Technology Exchange: | Bioinformatics, Computational biology, Crystallography, Mass spectrometry and Protein interaction |
| Keywords: | Adaptive Immunity, Autoimmunity and Crystallography |
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The process of structure solution: crystals, diffraction, electron density and molecular models. ...
Our group focuses on aspects of both innate and adaptive Immunity across species in an effort to understand the interplay between pathogens and specialized self molecules that initiate an immune response.
1. Understanding the Molecular Nature of Multiple Sclerosis and Autoimmunity
We study the molecules that are implicated in multiple sclerosis and have deciphered, in atomic detail, interactions between components of the human immune system and the infections that trigger relapses. Our results show that in a population susceptible for multiple sclerosis, common ailments ranging from the common cold to food poisoning have the potential to trick the immune system into attacking self tissue. We use both in vitro and in vivo tools to identify disease determinants. To look inside the microscopic world of the molecules of our body and understand their interactions, a machine the size of five football pitches called a synchrotron is used to shine high energy X-rays on biological crystals one billionth of a meter wide. The data we generate this way is used to create three dimensional reconstructions of the molecules implicated in immune response and we have so far shown that the basic molecular determinants that underpin disease onset is a handful of atoms (out of many thousands present) that look the same between a pathogen and human tissue. Our results indicate that although it is feasible to suggest who is likely to develop the disease in their lifetime (based on genetic screening), it becomes near impossible to predict if, when and how it will progress. However, having mapped the causative interactions, we are more likely to develop treatments that could target and mute those immune cells that are likely to promote disease.
2. Innate Immunity in Social Insects
The term Colony Collapse Disorder (CCD) describes the loss of bee hives due to the disappearance or death of mature worker bees leading to the depopulation and ultimate death of a honey bee colony. Although the causes of CCD are multi-factorial, environmental parameters uniformly appear to affect the efficiency of the honey bees’ immune responses, rendering individuals vulnerable to infections. In the honey bee, as in other insects and indeed higher vertebrates, an arsenal of specialized molecules is constitutively resident on or near the cell surface for the express purposes of recognizing and reacting to pathogens. However, we know very little of their structure or their interactions with other molecules.
We target Pathogen Pattern Recognition Receptors of the honey bee. We hope that by understanding how their immune system 'sees' pathogenic invasions we will contribute towards design of pharmaceuticals towards CCD control. We use recombinant full length proteins or domains therein which we further purify and crystallize. Diffraction-worthy crystals are then used to collect X-ray data and the resulting atomic models are compared against known structures that should allow us to usefully analyze the efficiency of aspects of the honey bee immune system.
| Name | Department | Institution | Country |
|---|---|---|---|
| Dr Astrid Iversen | Experimental Medicine Division | Oxford University | UK |
| Shirley Ellis | Institute for Animal Health, Compton | UK | |
| Petros Ligoxygakis | Biochemistry, University of Oxford | UK | |
| Stephan Feller | Weatherall Institute of Molecular Medicine | UK | |
| Pekka Pamilo | University of Helsinki | Finland |
2009. Distinct binding modes of two epitopes in Gab2 that interact with the SH3C domain of Grb2. Structure, 17 (6), pp. 809-822. Read abstract | Read more
Grb2 and Gab2 form a complex implicated in normal cell signaling and cancer development. Binding of the Grb2SH3C domain to Gab2 is essential for the interaction, but molecular details remained undefined. Using peptide arrays and isothermal titration calorimetry, two Grb2SH3C binding sites in Gab2 (Gab2a and Gab2b) were confirmed and characterized. Gab2a bears similarity to a p27Kip1 epitope that also binds Grb2SH3C. Crystal structures of both Gab2 epitopes complexed with Grb2SH3C reveal that Gab2b contains a 3(10) helix that positions the arginine and lysine of the core-binding motif RxxK in parallel orientation. In contrast, the Gab2a RxxK motif is embedded in a PPII helix with Arg and Lys in staggered orientation. A similar interaction mode is also present in a new complex of Mona/GadsSH3C with an RxxxxK epitope from the putative phosphatase HD-PTP. In summary, our study reveals interaction types of SH3 domains, highlighting their great versatility. Hide abstract
2007. Crystal structure and DNA repair activities of the AP endonuclease from Leishmania major. J Mol Biol, 373 (4), pp. 827-838. Read abstract | Read more
Apurinic/apyrimidinic endonucleases initiate the repair of abasic sites produced either spontaneously, from attack of bases by reactive oxygen species or as intermediates during base excision repair. The catalytic properties and crystal structure of Leishmania major apurinic/apyrimidinic endonuclease are described and compared with those of human APE1 and bacterial exonuclease III. The purified enzyme is shown to possess apurinic/apyrimidinic endonuclease activity of the same order as eukaryotic and prokaryotic counterparts and an equally robust 3'-phosphodiesterase activity. Consistent with this, expression of the L. major endonuclease confers resistance to both methyl methane sulphonate and H2O2 in Escherichia coli repair-deficient mutants while expression of the human homologue only reverts methyl methane sulphonate sensitivity. Structural analyses and modelling of the enzyme-DNA complex demonstrates a high degree of conservation to previously characterized homologues, although subtle differences in the active site geometry might account for the high 3'-phosphodiesterase activity. Our results confirm that the L. major's enzyme is a key element in mediating repair of apurinic/apyrimidinic sites and 3'-blocked termini and therefore must play an important role in the survival of kinetoplastid parasites after exposure to the highly oxidative environment within the host macrophage. Hide abstract
2006. The C-terminal SH3 domain of CRKL as a dynamic dimerization module transiently exposing a nuclear export signal. Structure, 14 (12), pp. 1741-1753. Read abstract | Read more
CRKL plays essential roles in cell signaling. It consists of an N-terminal SH2 domain followed by two SH3 domains. SH2 and SH3N bind to signaling proteins, but the function of the SH3C domain has remained largely enigmatic. We show here that the SH3C of CRKL forms homodimers in protein crystals and in solution. Evidence for dimer formation of full-length CRKL is also presented. In the SH3C dimer, a nuclear export signal (NES) is mostly buried under the domain surface. The same is true for a monomeric SH3C obtained under different crystallization conditions. Interestingly, partial SH3 unfolding, such as occurs upon dimer/monomer transition, produces a fully-accessible NES through translocation of a single beta strand. Our results document the existence of an SH3 domain dimer formed through exchange of the first SH3 domain beta strand and suggest that partial unfolding of the SH3C is important for the relay of information in vivo. Hide abstract
2006. The structure of the human allo-ligand HLA-B*3501 in complex with a cytochrome p450 peptide: steric hindrance influences TCR allo-recognition. Eur J Immunol, 36 (12), pp. 3288-3293. Read abstract | Read more
Virus-specific T cell populations have been implicated in allo-recognition. The subdominant T cell receptor JL12 recognizes both HLA-B*0801 presenting the Epstein-Barr virus-derived peptide FLRGRAYGL and also HLA-B*3501 presenting the cytochrome p450 self peptide KPIVVLHGY. This cross-reactivity could promote the rejection of HLA-B*3501-positive cells in Epstein-Barr virus-exposed HLA-B*0801 recipients. LC13, the dominant TCR against the HLA-B*0801:FLRGRAYGL complex, fails to recognize HLA-B*3501:KPIVVLHGY. We report the 1.75-Angstrom resolution crystal structure of the human allo-ligand HLA-B*3501:KPIVVLHGY. Similarities between this structure and that of HLA-B*0801:FLRGRAYGL may facilitate cross-recognition by JL12. Moreover, the elevated peptide position in HLA-B*3501:KPIVVLHGY would provide steric hindrance to LC13, preventing it from interacting in the manner in which it interacts with HLA-B*0801:FLRGRAYGL. These findings are relevant to understanding the basis of T cell cross-reactivity in allo-recognition, optimal transplant donor-recipient matching and developing specific molecular inhibitors of allo-recognition. Hide abstract
2004. Mona/Gads SH3C binding to hematopoietic progenitor kinase 1 (HPK1) combines an atypical SH3 binding motif, R/KXXK, with a classical PXXP motif embedded in a polyproline type II (PPII) helix. J Biol Chem, 279 (27), pp. 28724-28732. Read abstract | Read more
Hematopoietic progenitor kinase 1 (HPK1) is implicated in signaling downstream of the T cell receptor. Its non-catalytic, C-terminal half contains several prolinerich motifs, which have been shown to interact with different SH3 domain-containing adaptor proteins in vitro. One of these, Mona/Gads, was also shown to bind HPK1 in mouse T cells in vivo. The region of HPK1 that binds to the Mona/Gads C-terminal SH3 domain has been mapped and shows only very limited similarity to a recently identified high affinity binding motif in SLP-76, another T-cell adaptor. Using isothermal titration calorimetry and x-ray crystallography, the binding of the HPK1 motif to Mona/Gads SH3C has now been characterized in molecular detail. The results indicate that although charge interactions through an RXXK motif are essential for complex formation, a PXXP motif in HPK1 strongly complements binding. This unexpected binding mode therefore differs considerably from the previously described interaction of Mona/Gads SH3C with SLP-76. The crystal structure of the complex highlights the great versatility of SH3 domains, which allows interactions with very different proteins. This currently limits our ability to categorize SH3 binding properties by simple rules. Hide abstract
2004. The crystal structure of Trypanosoma cruzi dUTPase reveals a novel dUTP/dUDP binding fold. Structure, 12 (1), pp. 41-53. Read abstract | Read more
dUTPase is an essential enzyme involved with nucleotide metabolism and replication. We report here the X-ray structure of Trypanosoma cruzi dUTPase in its native conformation and as a complex with dUDP. These reveal a novel protein fold that displays no structural similarities to previously described dUTPases. The molecular unit is a dimer with two active sites. Nucleotide binding promotes extensive structural rearrangements, secondary structure remodeling, and rigid body displacements of 20 A or more, which effectively bury the substrate within the enzyme core for the purpose of hydrolysis. The molecular complex is a trapped enzyme-substrate arrangement which clearly demonstrates structure-induced specificity and catalytic potential. This enzyme is a novel dUTPase and therefore a potential drug target in the treatment of Chagas' disease. Hide abstract
2003. Structural basis for SH3 domain-mediated high-affinity binding between Mona/Gads and SLP-76. EMBO J, 22 (11), pp. 2571-2582. Read abstract | Read more
SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2-like adaptor protein Mona/Gads associates stably with the T-cell receptor signal transducer SLP-76. The crystal structure of a complex between the C-terminal SH3 domain (SH3C) of Mona/Gads and a SLP-76 peptide has now been solved to 1.7 A. The peptide lacks the canonical SH3 domain binding motif P-x-x-P and does not form a frequently observed poly-proline type II helix. Instead, it adopts a clamp-like shape around the circumfence of the SH3C beta-barrel. The central R-x-x-K motif of the peptide forms a 3(10) helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high affinity. Interestingly, the SH3C displays ion-dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 domain functions. Hide abstract
Innate Immunity in the Honey Bee - The case of Colony Collapse Disorder
Innate Immunity in the Honey Bee - The case of Colony Collapse Disorder (CCD) The term Colony Collapse Disorder describes the loss of hives due to the disappearance or death of mature worker bees leading to the depopulation and ultimate death of a honey bee colony. Over a quarter of hives in the USA were lost to CCD last year and equivalent losses were reported across continental Europe and Asia. If CCD incidence persists over the next years it could conceivably signal the loss of the major ...
The Molecular Roots of Multiple Sclerosis
When our Bodies Turn Against Themselves The immune system is a near perfect fighting machine that can recognise millions of pathogenic life forms. The foot soldiers in this battle are T lymphocytes which will either directly attack infected cells or signal the presence of invaders to antibody producing cells. They recognise infection via specialised T cell receptors (TCRs) which bind antigenic peptides presented by MHC molecules on the surface of infected cells. Through an intricate series of ...
Toward a functional and structural understanding of HIV specific CTL-responses.
The best hope for ending the human immunodeficiency virus (HIV-1) pandemic is the development of a safe and effective vaccine. However, HIV does not lend itself to traditional methods of vaccine development and a deeper understanding of the interplay between the virus and its human host is badly needed at present. This project aims to address this need and is a collaboration between the Iversen group (Human immunology, WIMM) and the Harkiolaki group (Structural Immunology, WTCHG). The ...
