Cryo-EM, crystallography and functional studies of human membrane proteins involved in cancer, metabolic and neuropsychiatric disease

Project Overview

We aim to understand the fundamental causes of diseases such as cancer, metabolic diseases, neuropsychiatric disorders such as autism, bipolar disease and schizophrenia, and inflammation. All of these diseases are caused by failure of cells to make proteins that are properly folded and functional. In the Carpenter lab we study a particularly challenging set of molecules, proteins that are embedded within the membranes of cells and organelles. There are now many studies in progress to identify the genes that are mutated in a range of diseases. Working with collaborators in academia and industry, we have selected a series of proteins that are mutated in cancer, or implicated in neuropsychiatric disorders, metabolic and inflammation related diseases. For each of these proteins we aim to produce the protein, develop assays to study their function and solve their structures.

Structural biology for challenging proteins such as membrane proteins and large complexes  has taken a huge leap forward in recent years with the availability of new techniques such as electron cryo-microscopy. We now have a hugely expanded tool kit to allow us to understand the structures of proteins and we use and adopt novel technologies in order to solve the structures of disease-related proteins. We have solved structures of seven human membrane proteins by crystallography and three by cryo-EM, including PKD2, an ion channel involved in polycystic kidney disease and TMEM16K, a lipid scramblase associated with the cerebellar ataxia SCAR10. We have access to several cryo-EM microscope facilities at STRUBI, the Dunn School, at the eBIC National Cryo-EM Faciliy and through our collaborations with colleagues in the Pharma industry. We work very closely with 10 Pharmaceutical companies in developing our programs, and we publish all our research without restriction and without taking out intellectual property rights, so that we can progress the science as fast as possible.

We have a series of projects involving human ion channels, solute carriers, ABC transporters and enzymes, which would provide an in-depth understanding of disease mechanisms in bipolar disease, schizophrenia, cancer and metabolic diseases. We would welcome enthusiastic and highly motivated students who are keen to take advantage of the new technologies to make a real difference for patients.

Training Opportunities

The student will gain a solid grounding in the emerging area of membrane protein structural biology, combined with a knowledge of membrane protein structure and function. They will take advantage of the cutting edge technologies such as electron microscopy, as well as gaining extensive experience in classical X-ray crystallography. They will learn how to product proteins in the insect cells/baculovirus and mammalian expression systems, methods for screening for high levels of expression and purification, membrane protein purification, crystallisation and EM grid preparation. They will collect data and be involved in solving structures of complexes and novel membrane proteins, using whichever technique best suits the problem at hand. There will also be opportunities to study protein function, develop assays and identify small molecule and antibody binders that affect function. Together with information will help us to understand how disease-associated mutations affect the function of a protein, providing new insights into disease biology. This work will be done both in house and through collaborations in Oxford, in other academia labs and with our Pharmaceutical company collaborators. The student will learn how to work as part of a large team, manage experiments, present data, as well as learning how to write research papers and a thesis.

Theme

Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology

Admissions

Project reference number: 226

Funding and admissions information

Supervisors

Name Department Institution Country Email
Professor Liz Carpenter Structural Genomics Consortium Oxford University, Old Road Campus Research Building GBR liz.carpenter@sgc.ox.ac.uk
Dr Ashley C.W. Pike SGC Oxford GBR ashley.pike@sgc.ox.ac.uk

Spear ED, Hsu ET, Nie L, Carpenter EP, Hrycyna CA, Michaelis S. 2018. missense mutations that cause progeroid diseases decrease prelamin A cleavage activity and/or protein stability. Dis Model Mech, 11 (7), Read abstract | Read more

The human zinc metalloprotease ZMPSTE24 is an integral membrane protein crucial for the final step in the biogenesis of the nuclear scaffold protein lamin A, encoded by After farnesylation and carboxyl methylation of its C-terminal CAAX motif, the lamin A precursor (prelamin A) undergoes proteolytic removal of its modified C-terminal 15 amino acids by ZMPSTE24. Mutations in or that impede this prelamin A cleavage step cause the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS), and the related progeroid disorders mandibuloacral dysplasia type B (MAD-B) and restrictive dermopathy (RD). Here, we report the development of a 'humanized yeast system' to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated missense mutations. All mutations show diminished prelamin A processing and fall into three classes, with defects in activity, protein stability or both. Notably, some ZMPSTE24 mutants can be rescued by deleting the E3 ubiquitin ligase Doa10, involved in endoplasmic reticulum (ER)-associated degradation of misfolded membrane proteins, or by treatment with the proteasome inhibitor bortezomib. This finding may have important therapeutic implications for some patients. We also show that ZMPSTE24-mediated prelamin A cleavage can be uncoupled from the recently discovered role of ZMPSTE24 in clearance of ER membrane translocon-clogged substrates. Together with the crystal structure of ZMPSTE24, this humanized yeast system can guide structure-function studies to uncover mechanisms of prelamin A cleavage, translocon unclogging, and membrane protein folding and stability. Hide abstract

Bradley AR, Echalier A, Fairhead M, Strain-Damerell C, Brennan P, Bullock AN, Burgess-Brown NA, Carpenter EP, Gileadi O, Marsden BD, Lee WH, Yue W, Bountra C, von Delft F. 2017. The SGC beyond structural genomics: redefining the role of 3D structures by coupling genomic stratification with fragment-based discovery. Essays Biochem., 61 (5), pp. 495-503. Read abstract | Read more

The ongoing explosion in genomics data has long since outpaced the capacity of conventional biochemical methodology to verify the large number of hypotheses that emerge from the analysis of such data. In contrast, it is still a gold-standard for early phenotypic validation towards small-molecule drug discovery to use probe molecules (or tool compounds), notwithstanding the difficulty and cost of generating them. Rational structure-based approaches to ligand discovery have long promised the efficiencies needed to close this divergence; in practice, however, this promise remains largely unfulfilled, for a host of well-rehearsed reasons and despite the huge technical advances spearheaded by the structural genomics initiatives of the noughties. Therefore the current, fourth funding phase of the Structural Genomics Consortium (SGC), building on its extensive experience in structural biology of novel targets and design of protein inhibitors, seeks to redefine what it means to do structural biology for drug discovery. We developed the concept of a Target Enabling Package (TEP) that provides, through reagents, assays and data, the missing link between genetic disease linkage and the development of usefully potent compounds. There are multiple prongs to the ambition: rigorously assessing targets' genetic disease linkages through crowdsourcing to a network of collaborating experts; establishing a systematic approach to generate the protocols and data that comprise each target's TEP; developing new, X-ray-based fragment technologies for generating high quality chemical matter quickly and cheaply; and exploiting a stringently open access model to build multidisciplinary partnerships throughout academia and industry. By learning how to scale these approaches, the SGC aims to make structures finally serve genomics, as originally intended, and demonstrate how 3D structures systematically allow new modes of druggability to be discovered for whole classes of targets. Hide abstract

Clausen MV, Jarerattanachat V, Carpenter EP, Sansom MSP, Tucker SJ. 2017. Asymmetric mechanosensitivity in a eukaryotic ion channel. Proc. Natl. Acad. Sci. U.S.A., 114 (40), pp. E8343-E8351. Read abstract | Read more

Living organisms perceive and respond to a diverse range of mechanical stimuli. A variety of mechanosensitive ion channels have evolved to facilitate these responses, but the molecular mechanisms underlying their exquisite sensitivity to different forces within the membrane remains unclear. TREK-2 is a mammalian two-pore domain (K2P) K channel important for mechanosensation, and recent studies have shown how increased membrane tension favors a more expanded conformation of the channel within the membrane. These channels respond to a complex range of mechanical stimuli, however, and it is uncertain how differences in tension between the inner and outer leaflets of the membrane contribute to this process. To examine this, we have combined computational approaches with functional studies of oppositely oriented single channels within the same lipid bilayer. Our results reveal how the asymmetric structure of TREK-2 allows it to distinguish a broad profile of forces within the membrane, and illustrate the mechanisms that eukaryotic mechanosensitive ion channels may use to detect and fine-tune their responses to different mechanical stimuli. Hide abstract

Wilkes M, Madej MG, Kreuter L, Rhinow D, Heinz V, De Sanctis S, Ruppel S, Richter RM, Joos F, Grieben M, Pike AC, Huiskonen JT, Carpenter EP, Kühlbrandt W, Witzgall R, Ziegler C. 2017. Molecular insights into lipid-assisted Ca regulation of the TRP channel Polycystin-2. Nat. Struct. Mol. Biol., 24 (2), pp. 123-130. Read abstract | Read more

Polycystin-2 (PC2), a calcium-activated cation TRP channel, is involved in diverse Ca signaling pathways. Malfunctioning Ca regulation in PC2 causes autosomal-dominant polycystic kidney disease. Here we report two cryo-EM structures of distinct channel states of full-length human PC2 in complex with lipids and cations. The structures reveal conformational differences in the selectivity filter and in the large exoplasmic domain (TOP domain), which displays differing N-glycosylation. The more open structure has one cation bound below the selectivity filter (single-ion mode, PC2), whereas multiple cations are bound along the translocation pathway in the second structure (multi-ion mode, PC2). Ca binding at the entrance of the selectivity filter suggests Ca blockage in PC2, and we observed density for the Ca-sensing C-terminal EF hand in the unblocked PC2 state. The states show altered interactions of lipids with the pore loop and TOP domain, thus reflecting the functional diversity of PC2 at different locations, owing to different membrane compositions. Hide abstract

Grieben M, Pike AC, Shintre CA, Venturi E, El-Ajouz S, Tessitore A, Shrestha L, Mukhopadhyay S, Mahajan P, Chalk R, Burgess-Brown NA, Sitsapesan R, Huiskonen JT, Carpenter EP. 2017. Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2). Nat. Struct. Mol. Biol., 24 (2), pp. 114-122. Read abstract | Read more

Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli. Hide abstract

Dong YY, Pike AC, Mackenzie A, McClenaghan C, Aryal P, Dong L, Quigley A, Grieben M, Goubin S, Mukhopadhyay S, Ruda GF, Clausen MV, Cao L, Brennan PE, Burgess-Brown NA, Sansom MS, Tucker SJ, Carpenter EP. 2015. K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac. Science, 347 (6227), pp. 1256-9. Read abstract | Read more

TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac. Hide abstract

Stansfeld PJ, Goose JE, Caffrey M, Carpenter EP, Parker JL, Newstead S, Sansom MS. 2015. MemProtMD: Automated Insertion of Membrane Protein Structures into Explicit Lipid Membranes. Structure, 23 (7), pp. 1350-61. Read abstract | Read more

There has been exponential growth in the number of membrane protein structures determined. Nevertheless, these structures are usually resolved in the absence of their lipid environment. Coarse-grained molecular dynamics (CGMD) simulations enable insertion of membrane proteins into explicit models of lipid bilayers. We have automated the CGMD methodology, enabling membrane protein structures to be identified upon their release into the PDB and embedded into a membrane. The simulations are analyzed for protein-lipid interactions, identifying lipid binding sites, and revealing local bilayer deformations plus molecular access pathways within the membrane. The coarse-grained models of membrane protein/bilayer complexes are transformed to atomistic resolution for further analysis and simulation. Using this automated simulation pipeline, we have analyzed a number of recently determined membrane protein structures to predict their locations within a membrane, their lipid/protein interactions, and the functional implications of an enhanced understanding of the local membrane environment of each protein. Hide abstract

Qiu W, Liesa M, Carpenter EP, Shirihai OS. 2015. ATP Binding and Hydrolysis Properties of ABCB10 and Their Regulation by Glutathione. PLoS ONE, 10 (6), pp. e0129772. Read abstract | Read more

ABCB10 (ATP binding cassette sub-family B10) is a mitochondrial inner-membrane ABC transporter. ABCB10 has been shown to protect the heart from the impact of ROS during ischemia-reperfusion and to allow for proper hemoglobin synthesis during erythroid development. ABC transporters are proteins that increase ATP binding and hydrolysis activity in the presence of the transported substrate. However, molecular entities transported by ABCB10 and its regulatory mechanisms are currently unknown. Here we characterized ATP binding and hydrolysis properties of ABCB10 by using the 8-azido-ATP photolabeling technique. This technique can identify potential ABCB10 regulators, transported substrates and amino-acidic residues required for ATP binding and hydrolysis. We confirmed that Gly497 and Lys498 in the Walker A motif, Glu624 in the Walker B motif and Gly602 in the C-Loop motif of ABCB10 are required for proper ATP binding and hydrolysis activity, as their mutation changed ABCB10 8-Azido-ATP photo-labeling. In addition, we show that the potential ABCB10 transported entity and heme precursor delta-aminolevulinic acid (dALA) does not alter 8-azido-ATP photo-labeling. In contrast, oxidized glutathione (GSSG) stimulates ATP hydrolysis without affecting ATP binding, whereas reduced glutathione (GSH) inhibits ATP binding and hydrolysis. Indeed, we detectABCB10 glutathionylation in Cys547 and show that it is one of the exposed cysteine residues within ABCB10 structure. In all, we characterize essential residues for ABCB10 ATPase activity and we provide evidence that supports the exclusion of dALA as a potential substrate directly transported by ABCB10. Last, we show the first molecular mechanism by which mitochondrial oxidative status, through GSH/GSSG, can regulate ABCB10. Hide abstract

Quigley A, Dong YY, Pike AC, Dong L, Shrestha L, Berridge G, Stansfeld PJ, Sansom MS, Edwards AM, Bountra C, von Delft F, Bullock AN, Burgess-Brown NA, Carpenter EP. 2013. The structural basis of ZMPSTE24-dependent laminopathies. Science, 339 (6127), pp. 1604-7. Read abstract | Read more

Mutations in the nuclear membrane zinc metalloprotease ZMPSTE24 lead to diseases of lamin processing (laminopathies), such as the premature aging disease progeria and metabolic disorders. ZMPSTE24 processes prelamin A, a component of the nuclear lamina intermediate filaments, by cleaving it at two sites. Failure of this processing results in accumulation of farnesylated, membrane-associated prelamin A. The 3.4 angstrom crystal structure of human ZMPSTE24 has a seven transmembrane α-helical barrel structure, surrounding a large, water-filled, intramembrane chamber, capped by a zinc metalloprotease domain with the catalytic site facing into the chamber. The 3.8 angstrom structure of a complex with a CSIM tetrapeptide showed that the mode of binding of the substrate resembles that of an insect metalloprotease inhibitor in thermolysin. Laminopathy-associated mutations predicted to reduce ZMPSTE24 activity map to the zinc metalloprotease peptide-binding site and to the bottom of the chamber. Hide abstract

Shintre CA, Pike AC, Li Q, Kim JI, Barr AJ, Goubin S, Shrestha L, Yang J, Berridge G, Ross J, Stansfeld PJ, Sansom MS, Edwards AM, Bountra C, Marsden BD, von Delft F, Bullock AN, Gileadi O, Burgess-Brown NA, Carpenter EP. 2013. Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states. Proc. Natl. Acad. Sci. U.S.A., 110 (24), pp. 9710-5. Read abstract | Read more

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters. Hide abstract

Newstead S, Drew D, Cameron AD, Postis VL, Xia X, Fowler PW, Ingram JC, Carpenter EP, Sansom MS, McPherson MJ, Baldwin SA, Iwata S. 2011. Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2. EMBO J., 30 (2), pp. 417-26. Read abstract | Read more

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di- and tri-peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT(So), a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand-bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide-binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT(So) represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2. Hide abstract

Berridge G, Chalk R, D'Avanzo N, Dong L, Doyle D, Kim JI, Xia X, Burgess-Brown N, Deriso A, Carpenter EP, Gileadi O. 2011. High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins. Anal. Biochem., 410 (2), pp. 272-80. Read abstract | Read more

We have developed a method for intact mass analysis of detergent-solubilized and purified integral membrane proteins using liquid chromatography-mass spectrometry (LC-MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2-5 μg of protein. The method is also compatible with our standard LC-MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment. Hide abstract

Carpenter EP, Beis K, Cameron AD, Iwata S. 2008. Overcoming the challenges of membrane protein crystallography. Curr. Opin. Struct. Biol., 18 (5), pp. 581-6. Read abstract | Read more

Membrane protein structural biology is still a largely unconquered area, given that approximately 25% of all proteins are membrane proteins and yet less than 150 unique structures are available. Membrane proteins have proven to be difficult to study owing to their partially hydrophobic surfaces, flexibility and lack of stability. The field is now taking advantage of the high-throughput revolution in structural biology and methods are emerging for effective expression, solubilisation, purification and crystallisation of membrane proteins. These technical advances will lead to a rapid increase in the rate at which membrane protein structures are solved in the near future. Hide abstract

Newstead S, Fowler PW, Bilton P, Carpenter EP, Sadler PJ, Campopiano DJ, Sansom MS, Iwata S. 2009. Insights into how nucleotide-binding domains power ABC transport. Structure, 17 (9), pp. 1213-22. Read abstract | Read more

The mechanism by which nucleotide-binding domains (NBDs) of ABC transporters power the transport of substrates across cell membranes is currently unclear. Here we report the crystal structure of an NBD, FbpC, from the Neisseria gonorrhoeae ferric iron uptake transporter with an unusual and substantial domain swap in the C-terminal regulatory domain. This entanglement suggests that FbpC is unable to open to the same extent as the homologous protein MalK. Using molecular dynamics we demonstrate that this is not the case: both NBDs open rapidly once ATP is removed. We conclude from this result that the closed structures of FbpC and MalK have higher free energies than their respective open states. This result has important implications for our understanding of the mechanism of power generation in ABC transporters, because the unwinding of this free energy ensures that the opening of these two NBDs is also powered. Hide abstract

Weyand S, Shimamura T, Yajima S, Suzuki S, Mirza O, Krusong K, Carpenter EP, Rutherford NG, Hadden JM, O'Reilly J, Ma P, Saidijam M, Patching SG, Hope RJ, Norbertczak HT, Roach PC, Iwata S, Henderson PJ, Cameron AD. 2008. Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter. Science, 322 (5902), pp. 709-13. Read abstract | Read more

The nucleobase-cation-symport-1 (NCS1) transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85-angstrom resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, 10 of which are arranged in two inverted repeats of five helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved, showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine transporter LeuT(Aa) and the galactose transporter vSGLT reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronized by the inverted repeat helices 3 and 8, providing the structural basis of the alternating access model for membrane transport. Hide abstract