Dr Gary Thompson
Gary Thompson graduated in Chemistry from the University of York in 1981 and completed his PhD on complexes between sugars and Actinides at the University of Reading with Prof David Rice in 1984. Following his PhD he helped run the UK High Field NMR Facility at the University of Warwick. Between 1995 and 2000 he worked with Prof. Sheena Radford on the structure and folding of the single electron transfer blue copper protein pseudoazurin using NMR. Following a period as a postdoc with Prof Steve Homans at the University of Leeds using NMR to study vATPase, mouse urinary protein and MS2 bacteriophage, he became a staff member of the NMR Facility at the University of Leeds. During this time he was closely involved in the running and maintenance of the NMR Facility, helped found the Reson8 NMR managers group and worked on a wide range of projects using NMR to study the large protein complex FusB:EFG, the ion channel P7 from Hepatitis C, and the amyloid proteins from the ß2m system including the pathway of amyloid formation.
In 2017 Gary joined the University of Kent as NMR Facility Manager where he is in charge of the day to day running of the Facility, development of new techniques and training of users. Gary is also currently a member of the CCPN (combined Collaborative Computing Project for NMR) executive committee which guides the provision of software and teaching.
ORCID ID: 0000-0001-9399-7636
Gary's research interests fall in 4 main strands;
NMR Structure calculation and sparse restraints. NMR is one of the three main structural tools used to understand the structure and dynamics of molecules. Gary has a wide ranging interest in using NMR and integrated structural biology techniques used to understand protein-protein and protein-ligand interactions. These include a focus on the use of sparse restraints when calculating NMR structures and led to the development of the XCamshift implementation of the Camshift forcefield in xplor-nih (https://github.com/locsmith/xcamshift). Gary has used sparse restraints (RDCs, PREs, sPRES & chemical shifts) to calculate the structure of the 70kDa FusB:EFG complex and hidden states of the ß2m amyloid system.
Software tools. Gary is experienced in software development and has built a very fast chemical shift forcefield in python/cython that works with xplor-nih (https://github.com/locsmith/xcamshift). Currently he is collaborating with the CCPN software group to implement a modernised version of the CCPN data-model (https://www2.ccpn.ac.uk/api-documentation/ccpnmr/ccpnmr2.2/python/doc/api.html) and its toolchain using the XTEND computer language (https://www.eclipse.org/xtend/) and eclipse / papyrus uml model and tool chain (https://www.eclipse.org/papyrus/). Gary also has an interest in the use of diagrammatic representations of product operators (a fundamental tool use for the understanding of NMR pulse sequences) and there integration with symbolic maths programs programmed in python.
Structure and Dynamics of the protein PCTA. PCTA is a trans membrane protein from pseudomonas aureginosa that detects food sources (amino acids) and activates the CHE pathway which causes chemotaxis towards food sources. The mode of trans membrane signalling in the PCTA system is still not clearly understood and we are using NMR dynamics measurements (1H-15N) and RDCs to study the sensor domain of the protein (29kDa) in solution. (Collaboration with Jose Ortega Roldan)
Metabolomics There is a strong interest in the use of metabolomics and lipidomics in the School and Gary is collaborating with Jose Orteg-Roldan, Mark Shepherd and Anastasios Tsaousis to implement both current and novel metabolomics and lipidomics methodologies. This includes an interest in the use of 13C NMR and labelling in lipidomics and metabolomics studies.
Collaborations By the nature of his research Gary collaborates with a wide range of groups, current collaborators include;
Jose Ortega-Roldan, University of Kent Ben Goult, University of Kent Anastasios Tsaousis, University of Kent Andrew Lawrence / Warren Group, University of Kent Richard Williamson, University of Kent Arnout Kalverda, University of Leeds Stephan Kerpinski, Univsersity of Leeds Jennifer Tomlinson, University of Leeds Stephan Kepinski, University of Leeds Wael Houssan, University of Aberdeen Ed Brooksbank / Geetern Vuister, University of Leicester / CCPN
Gary teaches advanced NMR theory at the Reson8 NMR week on an annual basis.
MSc-R project available for 2019/20
This project will implement exciting new methods for the fast calculation of protein structures by NMR at the BioNMR facility. During the project you will build a pipeline to calculate the structures of proteins and use it to determine structures of domains from the Talin 2 system (Ben Goult). The method will integrate nOes and chemical shifts using xplor-nih, aria2 and XCamshift. Previous experience with protein expression and programming (e.g. python) would be beneficial but are not required. Training in the use of NMR structural methods, protein expression and python programming will be provided, all of which are highly transferable skills. The project will be jointly managed by NMR (Gary Thompson) Goult and Ortega-Roldan Labs.
Additional research costs £1200
Sanfelice, D., Koss, H., Bunney, T., Thompson, G., Farrell, B., Katan, M. and Breeze, A. (2018). NMR backbone assignments of the tyrosine kinase domain of human fibroblast growth factor receptor 3 in apo state and in complex with inhibitor PD173074. Biomolecular NMR Assignments [Online] 12:231-235. Available at: https://doi.org/10.1007/s12104-018-9814-7.Fibroblast growth factors receptors (FGFR) are transmembrane protein tyrosine kinases involved in many cellular process,
including growth, differentiation and angiogenesis. Dysregulation of FGFR enzymatic activity is associated with developmental
disorders and cancers; therefore FGFRs have become attractive targets for drug discovery, with a number of agents
in late-stage clinical trials. Here, we present the backbone resonance assignments of FGFR3 tyrosine kinase domain in the
ligand-free form and in complex with the canonical FGFR kinase inhibitor PD173074. Analysis of chemical shift changes
upon inhibitor binding highlights a characteristic pattern of allosteric network perturbations that is of relevance for future
drug discovery activities aimed at development of conformationally-selective FGFR inhibitors.
Bunney, T., Inglis, A., Sanfelice, D., Farrell, B., Kerr, C., Thompson, G., Masson, G., Thiyagarajan, N., Svergun, D., Williams, R., Breeze, A. and Katan, M. (2018). Disease Variants of FGFR3 Reveal Molecular Basis for the Recognition and Additional Roles for Cdc37 in Hsp90 Chaperone System. Structure [Online] 26:446-458. Available at: https://doi.org/10.1016/j.str.2018.01.016.Receptor tyrosine kinase FGFR3 is involved in many
signaling networks and is frequently mutated in
developmental disorders and cancer. The Hsp90/
Cdc37 chaperone system is essential for function of
normal and neoplastic cells. Here we uncover the
mechanistic inter-relationships between these pro-
teins by combining approaches including NMR,
HDX-MS, and SAXS. We show that several disease-
linked mutations convert FGFR3 to a stronger client,
where the determinant underpinning client strength
involves an allosteric network through the N-lobe
and at the lobe interface. We determine the architec-
ture of the client kinase/Cdc37 complex and dem on-
strate, together with site-speci?c information, that
binding of Cdc37 to unrelated kinases induces a
common, extensive conformational remodeling of
the kinase N-lobe, beyond localized changes and in-
teractions within the binary complex. As further
shown for FGFR3, this processing by Cdc37 deacti-
vates the kinase and presents it, in a speci ?c orienta-
tion established in the complex, for direct recognition
Karamanos, T., Pashley, C., Kalverda, A., Thompson, G., Mayzel, M., Orekhov, V. and Radford, S. (2016). A population shift between sparsely populated folding intermediates determines amyloidogenicity. Journal of the American Chemical Society [Online] 138:6271-6280. Available at: http://dx.doi.org/10.1021/jacs.6b02464.The balance between protein folding and misfolding is a crucial determinant of amyloid assembly. Transient intermediates that are sparsely populated during protein folding have been identified as key players in amyloid aggregation. However, due to their ephemeral nature, structural characterization of these species remains challenging. Here, using the power of non-uniformly sampled NMR methods we investigate the folding pathway of amyloidogenic and non-amyloidogenic variants of ?2-microglobulin (?2m) in atomic detail. Despite folding via common intermediate states, we show that the decreased population of the ITrans state and population of a less stable, more dynamic species ablates amyloid formation by increasing the energy barrier for amyloid assembly. The results show that subtle changes in conformational dynamics can have a dramatic effect in determining whether a protein is amyloidogenic, without perturbation of the mechanism of protein folding.
Tomlinson, J., Thompson, G., Kalverda, A., Zhuravleva, A. and O’Neill, A. (2016). A target-protection mechanism of antibiotic resistance at atomic resolution: insights into FusB-type fusidic acid resistance. Scientific Reports [Online] 6:19524-19524. Available at: http://dx.doi.org/10.1038/srep19524.Antibiotic resistance in clinically important bacteria can be mediated by proteins that physically associate with the drug target and act to protect it from the inhibitory effects of an antibiotic. We present here the first detailed structural characterization of such a target protection mechanism mediated through a protein-protein interaction, revealing the architecture of the complex formed between the FusB fusidic acid resistance protein and the drug target (EF-G) it acts to protect. Binding of FusB to EF G induces conformational and dynamic changes in the latter, shedding light on the molecular mechanism of fusidic acid resistance.
Klein, T., Vajpai, N., Phillips, J., Davies, G., Holdgate, G., Phillips, C., Tucker, J., Norman, R., Scott, A., Higazi, D., Lowe, D., Thompson, G. and Breeze, A. (2015). Structural and dynamic insights into the energetics of activation loop rearrangement in FGFR1 kinase. Nature Communications [Online] 6. Available at: http://dx.doi.org/10.1038/ncomms8877.Protein tyrosine kinases differ widely in their propensity to undergo rearrangements of the N-terminal Asp- Phe-Gly motif of the activation loop, with some, including FGFR1 kinase, appearing refractory to this so called ‘DFG flip’. Recent inhibitor-bound structures have unexpectedly revealed FGFR1 for the first time in a ‘DFG-out’ state. Here, we use conformationally-selective inhibitors as chemical probes for interrogation of the structural and dynamic features that appear to govern the DFG flip in FGFR1. Our detailed structural and biophysical insights identify contributions from altered dynamics in distal elements, including the ?H helix towards the outstanding stability of the DFG-out complex with the inhibitor ponatinib. We conclude that the ?C-?4 loop and ‘molecular brake’ regions together impose a high energy barrier for this conformational rearrangement, and that this may have significance for maintaining autoinhibition in the non-phosphorylated basal state of FGFR1.
Karamanos, T., Kalverda, A., Thompson, G. and Radford, S. (2015). Mechanisms of amyloid formation revealed by solution NMR. Progress in Nuclear Magnetic Resonance Spectroscopy [Online] 2015:86-104. Available at: http://dx.doi.org/10.1016/j.pnmrs.2015.05.002.Amyloid fibrils are proteinaceous elongated aggregates involved in more than fifty human diseases. Recent advances in electron microscopy and solid state NMR have allowed the characterization of fibril structures to different extents of refinement. However, structural details about the mechanism of fibril formation remain relatively poorly defined. This is mainly due to the complex, heterogeneous and transient nature of the species responsible for assembly; properties that make them difficult to detect and characterize in structural detail using biophysical techniques. The ability of solution NMR spectroscopy to investigate exchange between multiple protein states, to characterize transient and low-population species, and to study high molecular weight assemblies, render NMR an invaluable technique for studies of amyloid assembly. In this article we review state-of-the-art solution NMR methods for investigations of: (a) protein dynamics that lead to the formation of aggregation-prone species; (b) amyloidogenic intrinsically disordered proteins; and (c) protein–protein interactions on pathway to fibril formation. Together, these topics highlight the power and potential of NMR to provide atomic level information about the molecular mechanisms of one of the most fascinating problems in structural biology.
Monteiro, D., Patel, V., Bartlett, C., Nozaki, S., Grant, T., Gowdy, J., Thompson, G., Kalverda, A., Snell, E., Niki, H., Pearson, A. and Webb, M. (2015). The structure of the PanD/PanZ protein complex reveals negative feedback regulation of pantothenate biosynthesis by coenzyme A. Chemistry and biology [Online] 22:492-503. Available at: http://dx.doi.org/10.1016/j.chembiol.2015.03.017.Coenzyme A (CoA) is an ubiquitous and essential cofactor, synthesized from the precursor pantothenate. Vitamin biosynthetic pathways are normally tightly regulated, including the pathway from pantothenate to CoA. However, no regulation of pantothenate biosynthesis has been identified. We have recently described an additional component in the pantothenate biosynthetic pathway, PanZ, which promotes the activation of the zymogen, PanD, to form aspartate ?-decarboxylase (ADC) in a CoA-dependent manner. Here we report the structure of PanZ in complex with PanD, which reveals the structural basis for the CoA dependence of this interaction and activation. In addition, we show that PanZ acts as a CoA-dependent inhibitor of ADC catalysis. This inhibitory effect can effectively regulate the biosynthetic pathway to pantothenate, and thereby also regulate CoA biosynthesis. This represents a previously unobserved mode of metabolic regulation whereby a cofactor-utilizing protein negatively regulates the biosynthesis of the same cofactor.
Hiruma-Shimizu, K., Shimizu, H., Thompson, G., Kalverda, A. and Patching, S. (2015). Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications. Molecular Membrane Biology [Online] 32:139-155. Available at: http://dx.doi.org/10.3109/09687688.2015.1125536.Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-?-D-glucoside (?-OG), n-dodecyl-?-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.
Sarell, C., Karamanos, T., White, S., Bunka, D., Kalverda, A., Thompson, G., Barker, A., Stockley, P. and Radford, S. (2014). Distinguishing closely related amyloid precursors using an RNA aptamer. The Journal of Biological Chemistry [Online] 289:26859-26871. Available at: http://dx.doi.org/10.1074/jbc.M114.595066.Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human ?2-microglobulin (h?2m) can co-polymerize with its N-terminally truncated variant (?N6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using h?2m and its amyloidogenic counterpart, ??6, we describe selection of a 2'F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds h?2m with an EC50 of ?200 nM. NMR spectroscopy was used to assign the (1)H-(15)N HSQC spectrum of the B6-h?2m complex, revealing that the aptamer binds to the face of h?2m containing the A, B, E, and D ?-strands. In contrast, binding of B6 to ?N6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of h?2m and ?N6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.
Morin, S., Linnet, T., Lescanne, M., Schanda, P., Thompson, G., Tollinger, M., Teilum, K., Gagne, S., Marion, D. and Griesinger, C. (2014). relax: the analysis of biomolecular kinetics and thermodynamics using NMR relaxation dispersion data. Bioinformatics [Online] 30:2219-2220. Available at: http://dx.doi.org/10.1093/bioinformatics/btu166.Nuclear magnetic resonance (NMR) is a powerful tool for observing the motion of biomolecules at the atomic level. One technique, the analysis of relaxation dispersion phenomenon, is highly suited for studying the kinetics and thermodynamics of biological processes. Built on top of the relax computational environment for NMR dynamics is a new dispersion analysis designed to be comprehensive, accurate and easy-to-use. The software supports more models, both numeric and analytic, than current solutions. An automated protocol, available for scripting and driving the graphical user interface (GUI), is designed to simplify the analysis of dispersion data for NMR spectroscopists. Decreases in optimization time are granted by parallelization for running on computer clusters and by skipping an initial grid search by using parameters from one solution as the starting point for another -using analytic model results for the numeric models, taking advantage of model nesting, and using averaged non-clustered results for the clustered analysis.
Foster, T., Thompson, G., Kalverda, A., Kankanala, J., Bentham, M., Wetherill, L., Thompson, J., Barker, A., Clarke, D., Noerenberg, M., Pearson, A., Rowlands, D., Homans, S., Harris, M., Foster, R. and Griffin, S. (2014). Structure-guided design affirms inhibitors of hepatitis C virus p7 as a viable class of antivirals targeting virion release. Hepatology [Online] 59:408-422. Available at: http://dx.doi.org/10.1002/hep.26685.Current interferon-based therapy for hepatitis C virus (HCV) infection is inadequate, prompting a shift toward combinations of direct-acting antivirals (DAA) with the first protease-targeted drugs licensed in 2012. Many compounds are in the pipeline yet primarily target only three viral proteins, namely, NS3/4A protease, NS5B polymerase, and NS5A. With concerns growing over resistance, broadening the repertoire for DAA targets is a major priority. Here we describe the complete structure of the HCV p7 protein as a monomeric hairpin, solved using a novel combination of chemical shift and nuclear Overhauser effect (NOE)-based methods. This represents atomic resolution information for a full-length virus-coded ion channel, or “viroporin,” whose essential functions represent a clinically proven class of antiviral target exploited previously for influenza A virus therapy. Specific drug-protein interactions validate an allosteric site on the channel periphery and its relevance is demonstrated by the selection of novel, structurally diverse inhibitory small molecules with nanomolar potency in culture. Hit compounds represent a 10,000-fold improvement over prototypes, suppress rimantadine resistance polymorphisms at submicromolar concentrations, and show activity against other HCV genotypes. Conclusion: This proof-of-principle that structure-guided design can lead to drug-like molecules affirms p7 as a much-needed new target in the burgeoning era of HCV DAA.
Karamanos, T., Kalverda, A., Thompson, G. and Radford, S. (2014). Visualization of transient protein-protein interactions that promote or inhibit amyloid assembly. Molecular Cell [Online] 55:214-226. Available at: http://dx.doi.org/10.1016/j.molcel.2014.05.026.In the early stages of amyloid formation, heterogeneous populations of oligomeric species are generated, the affinity, specificity, and nature of which may promote, inhibit, or define the course of assembly. Despite the importance of the intermolecular interactions that initiate amyloid assembly, our understanding of these events remains poor. Here, using amyloidogenic and nonamyloidogenic variants of ?-microglobulin, we identify the interactions that inhibit or promote fibril formation in atomic detail. The results reveal that different outcomes of assembly result from biomolecular interactions involving similar surfaces. Specifically, inhibition occurs via rigid body docking of monomers in a head-to-head orientation to form kinetically trapped dimers. By contrast, the promotion of fibrillation involves relatively weak protein association in a similar orientation, which results in conformational changes in the initially nonfibrillogenic partner. The results highlight the complexity of interactions early in amyloid assembly and reveal atomic-level information about species barriers in amyloid formation.
Kalverda, A., Gowdy, J., Thompson, G., Homans, S., Henderson, P. and Patching, S. (2014). TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor. Molecular Membrane Biology [Online] 31:131-140. Available at: http://dx.doi.org/10.3109/09687688.2014.911980.Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [(15)N-(1)H]- and [(13)C-(1)H]-methyl-TROSY NMR spectra with a 52?kDa membrane protein that putatively has 12 transmembrane-spanning ?-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48?h at a temperature of 25?°C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan (15)N backbone positions and also resolved signals for (15)N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [(13)C-(1)H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex ?-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.
Czajlik, A., Thompson, G., Khan, G., Kalverda, A., Homans, S. and Trinick, J. (2014). ¹H, ¹?N, and ¹³C backbone chemical shift assignment of titin domains A59-A60 and A60 alone. Biomolecular NMR assignments [Online] 8:429-433. Available at: http://dx.doi.org/10.1007/s12104-013-9532-0.The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is > 1 ?m long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or 'super-repeats'. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619-628, 2012). Here we report backbone (15)N, (13)C and (1)H chemical shift and (13)C? assignments for the A59-A60 domain tandem from the titin A59-A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59-A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59-A60.
Czajlik, A., Thompson, G., Khan, G., Kalverda, A., Homans, S. and Trinick, J. (2014). 1H, 15N and 13C backbone chemical shift assignment of the titin A67-A68 domain tandem. Biomolecular NMR Assignments [Online] 6:39-41. Available at: http://dx.doi.org/10.1007/s12104-011-9321-6.The giant protein titin is the third most abundant protein of vertebrate striated muscle. The titin molecule is >1 ?m long and spans half the sarcomere, from the Z-disk to the M-line, and has important roles in sarcomere assembly, elasticity and intracellular signaling. In the A-band of the sarcomere titin is attached to the thick filaments and mainly consists immunoglobulin-like and fibronectin type III-like domains. These are mostly arranged in long-range patterns or ‘super-repeats’. The large super-repeats each contain 11 domains and are repeated 11 times, thus forming nearly half the titin molecule. Through interactions with myosin and C-protein, they are involved in thick filament assembly. The importance of titin in muscle assembly is highlighted by the effect of mutations in the A-band portion, which are the commonest cause of dilated cardiomyopathy, affecting ~1 in 250 (Herman et al. in N Engl J Med 366:619–628, 2012). Here we report backbone 15N, 13C and 1H chemical shift and 13C? assignments for the A59–A60 domain tandem from the titin A59–A69 large super-repeat, completed using triple resonance NMR. Since, some regions of the backbone remained unassigned in A60 domain of the complete A59–A60 tandem, a construct containing a single A60 domain, A60sd, was also studied using the same methods. Considerably improved assignment coverage was achieved using A60sd due to its lower mass and improved molecular tumbling rate; these assignments also allowed the analysis of inter-domain interactions using chemical shift mapping against A59–A60.
Pashley, C., Morgan, G., Kalverda, A., Thompson, G., Kleanthous, C. and Radford, S. (2012). Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions. Journal of Molecular Biology [Online] 416:300-318. Available at: http://dx.doi.org/10.1016/j.jmb.2011.12.041.The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A-L19A-L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, (15)N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A-L19A-L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (R(h)=25 Å) relative to the urea-denatured state (R(h)?30 Å) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state.
Cox, G., Thompson, G., Jenkins, H., Peske, F., Savelsbergh, A., Rodnina, M., Wintermeyer, W., Homans, S., Edwards, T. and O’Neill, A. (2012). Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid. Proceedings of the National Academy of Sciences [Online] 109:2102-2107. Available at: https://doi.org/10.1073/pnas.1117275109.Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusB-type proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FA-mediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. This domain mediates a high-affinity interaction with the C-terminal domains of EF-G. By binding to EF-G on the ribosome, FusB-type proteins promote the dissociation of stalled ribosome?EF-G?GDP complexes that form in the presence of FA, thereby allowing the ribosomes to resume translation. Ribosome clearance by these proteins represents a highly unusual antibiotic resistance mechanism, which appears to be fine-tuned by the relative abundance of FusB-type protein, ribosomes, and EF-G.
Heyda, J., Koz?i?s?ek, M., Bedna?rova, L., Thompson, G., Konvalinka, J., Vondra?s?ek, J. and Jungwirth, P. (2011). Urea and Guanidinium Induced Denaturation of a Trp-Cage Miniprotein. The Journal of Physical Chemistry B [Online] 115:8910-8924. Available at: http://dx.doi.org/10.1021/jp200790h.Using a combination of experimental techniques (circular dichroism, differential scanning calorimetry, and NMR) and molecular dynamics simulations, we performed an extensive study of denaturation of the Trp-cage miniprotein by urea and guanidinium. The experiments, despite their different sensitivities to various aspects of the denaturation process, consistently point to simple, two-state unfolding process. Microsecond molecular dynamics simulations with a femtosecond time resolution allow us to unravel the detailed molecular mechanism of Trp-cage unfolding. The process starts with a destabilizing proline shift in the hydrophobic core of the miniprotein, followed by a gradual destruction of the hydrophobic loop and the ?-helix. Despite differences in interactions of urea vs guanidinium with various peptide moieties, the overall destabilizing action of these two denaturants on Trp-cage is very similar.
Eichner, T., Kalverda, A., Thompson, G., Homans, S. and Radford, S. (2011). Conformational Conversion during Amyloid Formation at Atomic Resolution. Molecular Cell [Online] 41:161-172. Available at: http://dx.doi.org/10.1016/j.molcel.2010.11.028.Numerous studies of amyloid assembly have indicated that partially folded protein species are responsible for initiating aggregation. Despite their importance, the structural and dynamic features of amyloidogenic intermediates and the molecular details of how they cause aggregation remain elusive. Here, we use ?N6, a truncation variant of the naturally amyloidogenic protein ?(2)-microglobulin (?(2)m), to determine the solution structure of a nonnative amyloidogenic intermediate at high resolution. The structure of ?N6 reveals a major repacking of the hydrophobic core to accommodate the nonnative peptidyl-prolyl trans-isomer at Pro32. These structural changes, together with a concomitant pH-dependent enhancement in backbone dynamics on a microsecond-millisecond timescale, give rise to a rare conformer with increased amyloidogenic potential. We further reveal that catalytic amounts of ?N6 are competent to convert nonamyloidogenic human wild-type ?(2)m (H?(2)m) into a rare amyloidogenic conformation and provide structural evidence for the mechanism by which this conformational conversion occurs.
Houssen, W., Wright, S., Kalverda, A., Thompson, G., Kelly, S. and Jaspars, M. (2010). Solution Structure of the Leader Sequence of the Patellamide Precursor Peptide, PatE1-34. ChemBioChem [Online] 11:1867-1873. Available at: http://dx.doi.org/10.1002/cbic.201000305.The solution structure of the leader sequence of the patellamide precursor peptide was analysed by using CD and determined with NOE-restrained molecular dynamics calculations. This leader sequence is highly conserved in the precursor peptides of some other cyanobactins harbouring heterocycles, and is assumed to play a role in targeting the precursor peptide to the post-translational machinery. The sequence was observed to form an alpha-helix spanning residues 13-28 with a hydrophobic surface on one side of the helix. This hydrophobic surface is proposed to be the site of the initial binding with modifying enzymes.
Kalverda, A., Thompson, G., Vogel, A., Schröder, M., Bowie, A., Khan, A. and Homans, S. (2009). Poxvirus K7 Protein Adopts a Bcl-2 Fold: Biochemical Mapping of Its Interactions with Human DEAD Box RNA Helicase DDX3. Journal of Molecular Biology [Online] 385:843-853. Available at: http://dx.doi.org/10.1016/j.jmb.2008.09.048.Poxviruses have evolved numerous strategies to evade host innate immunity. Vaccinia virus K7 is a 149-residue protein with previously unknown structure that is highly conserved in the orthopoxvirus family. K7 bears sequence and functional similarities to A52, which interacts with interleukin receptor-associated kinase 2 and tumor necrosis factor receptor-associated factor 6 to suppress nuclear factor kappaB activation and to stimulate the secretion of the anti-inflammatory cytokine interleukin-10. In contrast to A52, K7 forms a complex with DEAD box RNA helicase DDX3, thereby suppressing DDX3-mediated ifnb promoter induction. We determined the NMR solution structure of K7 to provide insight into the structural basis for poxvirus antagonism of innate immune signaling. The structure reveals an alpha-helical fold belonging to the Bcl-2 family despite an unrelated primary sequence. NMR chemical-shift mapping studies have localized the binding surface for DDX3 on a negatively charged face of K7. Furthermore, thermodynamic studies have mapped the K7-binding region to a 30-residue N-terminal fragment of DDX3, ahead of the core RNA helicase domains.
Rolfsson, O., Toropova, K., Morton, V., Francese, S., Basnak, G., Thompson, G., Homans, S., Ashcroft, A., Stonehouse, N. and Ranson, N. (2008). RNA Packing Specificity and Folding during Assembly of the Bacteriophage MS2. Computational and Mathematical Methods in Medicine [Online] 9:339-349. Available at: http://dx.doi.org/10.1080/17486700802168445.Using a combination of biochemistry, mass spectrometry, NMR spectroscopy and cryo-electron microscopy (cryo-EM), we have been able to show that quasi-equivalent conformer switching in the coat protein (CP) of an RNA bacteriophage (MS2) is controlled by a sequence-specific RNA–protein interaction. The RNA component of this complex is an RNA stem-loop encompassing just 19 nts from the phage genomic RNA, which is 3569 nts in length. This binding results in the conversion of a CP dimer from a symmetrical conformation to an asymmetric one. Only when both symmetrical and asymmetrical dimers are present in solution is assembly of the T?=?3 phage capsid efficient. This implies that the conformers, we have characterized by NMR correspond to the two distinct quasi-equivalent conformers seen in the 3D structure of the virion. An icosahedrally-averaged single particle cryo-EM reconstruction of the wild-type phage (to ?9?Å resolution) has revealed icosahedrally ordered density encompassing up to 90% of the single-stranded RNA genome. The RNA is seen with a novel arrangement of two concentric shells, with connections between them along the 5-fold symmetry axes. RNA in the outer shell interacts with each of the 90 CP dimers in the T?=?3 capsid and although the density is icosahedrally averaged, there appears to be a different average contact at the different quasi-equivalent protein dimers: precisely the result that would be expected if protein conformer switching is RNA-mediated throughout the assembly pathway. This unprecedented RNA structure provides new constraints for models of viral assembly and we describe experiments aimed at probing these. Together, these results suggest that viral genomic RNA folding is an important factor in efficient assembly, and further suggest that RNAs that could sequester viral CPs but not fold appropriately could act as potent inhibitors of viral assembly.
Sto?ckmann, H., Bronowska, A., Syme, N., Thompson, G., Kalverda, A., Warriner, S. and Homans, S. (2008). Residual Ligand Entropy in the Binding of p -Substituted Benzenesulfonamide Ligands to Bovine Carbonic Anhydrase II. Journal of the American Chemical Society [Online] 130:12420-12426. Available at: http://dx.doi.org/10.1021/ja803755m.In studies on the thermodynamics of ligand-protein interactions, it is often assumed that the configurational and conformational entropy of the ligand is zero in the bound state (i.e., the ligand is rigidly fixed in the binding pocket). However, there is little direct experimental evidence for this assumption, and in the case of binding of p-substituted benzenesulfonamide inhibitors to bovine carbonic anhydrase II (BCA II), the observed thermodynamic binding signature derived from isothermal titration calorimetry experiments leads indirectly to the conclusion that a considerable degree of residual entropy remains in the bound ligand. Specifically, the entropy of binding increases with glycine chain length n, and strong evidence exists that this thermodynamic signature is not driven by solvent reorganization. By use of heteronuclear (15)N NMR relaxation measurements in a series (n = 1-6) of (15)N-glycine-enriched ligands, we find that the observed thermodynamic binding signature cannot be explained by residual ligand dynamics in the bound state, but rather results from the indirect influence of ligand chain length on protein dynamics.
Stockley, P., Rolfsson, O., Thompson, G., Basnak, G., Francese, S., Stonehouse, N., Homans, S. and Ashcroft, A. (2007). A simple, RNA-mediated allosteric switch controls the pathway to formation of a T=3 viral capsid. Journal of Molecular Biology [Online] 369:541-552. Available at: http://dx.doi.org/10.1016/j.jmb.2007.03.020.Using mass spectrometry we have detected both assembly intermediates and the final product, the T = 3 viral capsid, during reassembly of the RNA bacteriophage MS2. Assembly is only efficient when both types of quasiequivalent coat protein dimer seen in the final capsid are present in solution. NMR experiments confirm that interconversion of these conformers is allosterically regulated by sequence-specific binding of a short RNA stem-loop. Isotope pulse-chase experiments confirm that all intermediates observed are competent for further coat protein addition, i.e., they are all on the pathway to capsid formation, and that the unit of capsid growth is a coat protein dimer. The major intermediate species are dominated by stoichiometries derived from formation of the particle threefold axis, implying that there is a defined pathway toward the T=3 shell. These results provide the first experimental evidence for a detailed mechanistic explanation of the regulation of quasiequivalent capsid assembly. They suggest a direct role for the encapsidated RNA in assembly in vivo, which is consistent with the structure
Yung, A., Turnbull, W., Kalverda, A., Thompson, G., Homans, S., Kitov, P. and Bundle, D. (2003). Large-Scale Millisecond Intersubunit Dynamics in the B Subunit Homopentamer of the Toxin Derived from Escherichia coli O157. Journal of the American Chemical Society [Online] 125:13058-13062. Available at: http://dx.doi.org/10.1021/ja0367288.We report here solution NMR relaxation measurements that show millisecond time-scale intersubunit dynamics in the homopentameric B subunit (VTB) of the toxin derived from Escherichia coli O157. These data are consistent with interconversion between an axially symmetric form and a low-abundance (?10%, 45 °C) higher energy form. The higher energy state is depopulated on binding of a novel bivalent analogue (Pk dimer) of the natural carbohydrate acceptor globotriaosylceramide. The isothermal titration calorimetry isotherm for the binding of Pk dimer to VTB is consistent with a five-site sequential binding model which assumes that cooperative effects arise through communication only between neighboring binding sites. The resulting thermodynamic parameters (Ka1 = 114 ± 2.2 M-1, Ka2 = 283 ± 4.5 M-1, ?H1° = ?116.3 ± 0.55 kJ/mol, and ?H2° = ?50.3 ± 0.11 kJ/mol) indicate favorable entropic cooperativity that has not previously been observed in multivalent systems.
Milne, B., Morris, L., Jaspars, M. and Thompson, G. (2002). Conformational change in the thiazole and oxazoline containing cyclic octapeptides, the patellamides. Part 2. Solvent dependent conformational change. Journal of the Chemical Society, Perkin Transactions 2 [Online]:1076-1080. Available at: http://dx.doi.org/10.1039/b201824c.Solvent dependent conformational change of the thiazole and oxazoline containing cyclic peptides, the patellamides, is examined by a combination of experimental and theoretical methods. A mechanism for the simultaneous formation of two type-II ?-turns in the patellamides is proposed based on molecular dynamics and NOE restrained molecular dynamics studies as well as literature evidence. The effect of the solvent and desymmetrisation of the patellamides is crucial, with symmetrical patellamides in polar solvents giving the open type-I conformation, whereas symmetrical patellamides in non-polar solvents and asymmetrical patellamides in both polar and non-polar solvents give rise to the folded type-II conformation.
Morris, L., Milne, B., Thompson, G. and Jaspars, M. (2002). Conformational change in the thiazole and oxazoline containing cyclic octapeptides, the patellamides. Part 1. Cu2+ and Zn2+ induced conformational change. JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 2 [Online]:1072-1075. Available at: http://dx.doi.org/10.1039/b202823n.Conformational change during the binding of Cu2+ and Zn2+ to the thiazole and oxazoline containing cyclic octapeptides, the patellamides, is examined by a combination of experimental and theoretical methods. Circular dichroism and NOE-restrained molecular dynamics studies indicate that upon complexing with one equivalent of Cu2+, patellamide C undergoes a change in conformation which pre-organises a second Cu2+ binding site, and that the binding of a second Cu2+ induces no further conformational change. The binding of Zn2+ induces little conformational change in patellamide C. A restrained conformational search shows that the conformational change induced by the addition of one equivalent of Cu2+ to patellamide C is an intrinsic design feature of the system. Electronic structure calculations indicate that the patellamides provide an ideal coordination environment for Cu2+. On the basis of the evidence gathered, it can be proposed that Cu2+ is the biologically relevant metal for the patellamides.
Reader, J., van Nuland, N., Thompson, G., Ferguson, S., Dobson, C. and Radford, S. (2001). A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding. Protein Science [Online] 10:1216-1224. Available at: https://doi.org/10.1110/ps.52801.The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.
Thompson, G., Shimizu, H., Homans, S. and Donohue-Rolfe, A. (2000). Localization of the Binding Site for the Oligosaccharide Moiety of Gb 3 on Verotoxin 1 Using NMR Residual Dipolar Coupling Measurements. Biochemistry [Online] 39:13153-13156. Available at: http://dx.doi.org/10.1021/bi001394+.By use of NMR residual dipolar coupling measurements in a dilute liquid-crystalline solvent, the solution structure has been determined of the complex between the oligosaccharide moiety of globotriaosylceramide (Gb3-OS) and the B-subunit homopentamer of verotoxin 1 (VTB). The dipolar coupling data indicate that Gb3-OS binds in a single binding site per monomer, which is identical to one of three sites inferred from the X-ray structure of the same complex. We find no evidence within experimental error for occupancy at either of the two additional binding sites observed per monomer in the crystal structure.
Morris, L., Jantina Kettenes van den Bosch, J., Versluis, K., Thompson, G. and Jaspars, M. (2000). Structure Determination and MSn Analysis of Two New Lissoclinamides Isolated from the Indo–Pacific Ascidian Lissoclinum patella: NOE Restrained Molecular Dynamics Confirms the Absolute Stereochemistry Derived by Degradative Methods. Tetrahedron [Online] 56:8345-8353. Available at: http://dx.doi.org/10.1016/S0040-4020(00)00746-8.Two new lissoclinamides, lissoclinamides 9 and 10 were isolated from an Indonesian collection of the ascidian Lissoclinum patella along with the known patellamide C. The structures of the lissoclinamides were determined by a combination of 2D NMR, selective 1D TOCSY, MS and MSn techniques. The assignment of absolute stereochemistry was achieved by the hydrolysis of lissoclinamides 9 and 10 followed by chiral TLC. In the case of lissoclinamide 9, NOE restrained molecular dynamics studies were also performed confirming the proposed stereochemistry.
Thompson, G., Radford, S., Leung, Y., Redfield, C. and Ferguson, S. (2000). The structure and dynamics in solution of Cu(I) pseudoazurin from Paracoccus pantotrophus. Protein Science [Online] 9:846-858. Available at: http://dx.doi.org/10.1110/ps.9.5.846.The solution structure and backbone dynamics of Cu(I) pseudoazurin, a 123 amino acid electron transfer protein from Paracoccus pantotrophus, have been determined using NMR methods. The structure was calculated to high precision, with a backbone RMS deviation for secondary structure elements of 0.35+/-0.06 A, using 1,498 distance and 55 torsion angle constraints. The protein has a double-wound Greek-key fold with two alpha-helices toward its C-terminus, similar to that of its oxidized counterpart determined by X-ray crystallography. Comparison of the Cu(I) solution structure with the X-ray structure of the Cu(II) protein shows only small differences in the positions of some of the secondary structure elements. Order parameters S2, measured for amide nitrogens, indicate that the backbone of the protein is rigid on the picosecond to nanosecond timescale.
thompson, g and kalvderda, a (2013). Tools and Techniques in Biomolecular Science. In: divan, aysha and royds, janice eds. Tools and Techniques in Biomolecular Science. Oxford University Press. Available at: https://doi.org/ISBN: 9780199695560.
Kalverda, A., Thompson, G. and Turnbull, W. (2005). The importance of protein structural dynamics and the contribution of NMR spectroscopy. In: Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics. John Wiley and Sons, pp. 2578-2582.