Gordon Roberts Research Interests

Recent Publications

The biological functions of proteins depend on their ability to bind other molecules with remarkable specificity. Thus, for example, enzymes must recognise their substrates, and cellular regulation depends on a cascade of specific protein-protein and protein-nucleic acid interactions. A major interest of our group is to reach a detailed understanding of these recognition processes, particularly in the contexts of protein-protein interactions (in biological electron transfer and in cell signalling) and enzyme mechanisms.

The first essential is to know the structure of the interacting molecules. Our structural tools include nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and small-angle X-ray and neutron scattering (SAXS and SANS). By combining structural studies with varying the structure of the protein and its binding partner(s), using the techniques of protein engineering, we can build up a detailed picture of the contributions of individual interactions to the recognition process and to the function of the protein. It is becoming widely recognised that many - perhaps most - of these specific recognition processes involve changes in protein conformation, and this has been a particularly important focus of our work.

Over the last few years our work has primarily been in three areas:

• Inhibitor binding to metallo-β-lactamases
The rapid increase in resistance to β-lactam antibiotics is a major clinical and public health concern, as these antibiotics have long been key to the treatment of serious bacterial infections. The β-lactamases, enzymes which inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond, are a major source of this resistance. In the sequence-based classification of β-lactamases, classes A, C and D are serine enzymes, while class B represents the zinc dependent metallo-β-lactamase enzymes (MBLs); the latter are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics. Transferable MBLs have a worldwide spread and are encountered in several gram-negative pathogenic bacteria and the occurrence of new transferable MBLs such as NDM-1 is an indicator of a serious, evolving and escalating problem.

By contrast with the success in developing inhibitors of serine β-lactamases such as clavulanic acid, no clinically efficient inhibitors of MBLs have yet been reported. While all MBLs share conserved motifs, the sequence similarity is low and the range of active site architectures makes the development of broad spectrum inhibitors difficult. We have identified R-thiomandelate as a promising reasonably broad spectrum inhibitor of MBLs, combining a thiol functionality which binds simultaneously to the two zinc atoms with a carboxylate function which increases its inhibitory potency.

We have determined the high resolution solution NMR structures of the Bacillus cereus metallo-β-lactamase, BcII, and of its complex with R-thiomandelic acid. This is the first reported solution structure of any metallo-β-lactamase. There are differences between the solution structure of the free enzyme and previously reported crystal structures in the loops flanking the active site, which are important for substrate and inhibitor binding and catalysis. Changes in the enzyme structure upon inhibitor binding clarify the role of the mobile β3-β4 loop. Comparisons with other metallo-β-lactamases highlight the roles of individual amino-acid residues in the active site and the β3-β4 loop in inhibitor binding and provide information on the basis of structure-activity relationships among metallo-β-lactamase inhibitors. 

Selected publications on metallo-β-lactamases:

  • Mollard, C., Moali, C., Papamicael, C., Damblon, C., Vessilier, S., Amicosante, G., Schofield, C. J., Galleni, M., Frère, J.-M., and Roberts, G.C.K. (2001) Thiomandelic acid, a broad-spectrum inhibitor of zinc β-lactamases: kinetic and spectroscopic studies. J. Biol. Chem., 276, 45015-45023. doi:10.1074/jbc.M107054200
  • Damblon, C.F., Jensen, M., Ababou, A., Barsukov, I., Papamicael, C., Schofield, C.J., Olsen, L., Bauer, R., and Roberts, G.C.K. (2003) The inhibitor thiomandelic acid binds to both metal ions in metallo-β-lactamase and induces positive cooperativity in metal binding. J. Biol. Chem., 278, 29240 – 29251. doi:10.1074/jbc.M301562200
  • Jacquin, O., Balbeur, D., Damblon, C., Marchot, P., De Pauw, E., Roberts, G.C.K., Frère, J.-M., and Matagne, A. (2009) Positively cooperative binding of zinc ions to Bacillus cereus 569/H/9 β-lactamase II suggests that the binuclear enzyme is the only relevant form for catalysis. J. Mol. Biol. 392, 1278–1291. doi:10.1016/j.jmb.2009.097.02
  • Karsisiotis, A.I., Damblon, C., and Roberts, G.C.K. (2013) Solution structures of the Bacillus cereus metallo-β-lactamase BcII and its complex with the inhibitor R-thiomandelic acid. Biochem. J., 456, 397-407. doi:10.1042/BJ20131003 

• Proteins involved in Integrin-Mediated Cell Adhesion.
Integrins are a large family of cell surface transmembrane adhesion receptors that mediate both cell-cell and cell-extracellular matrix (ECM) interactions. They are heterodimers of α and β subunits, each containing a large extracellular domain (~80-150 kDa), a single transmembrane α-helix and a cytoplasmic domain or ‘tail’ of 10-70 residues. Cell adhesion to the ECM is fundamental to the development of multi-cellular organisms, and involves the coordinated assembly and disassembly of integrins into complexes called focal adhesions. In these complexes, the internal tails of integrin β-subunits are typically linked to the actin cytoskeleton via cytoplasmic proteins with scaffolding, adaptor, regulatory and mechano-transduction functions. Among these proteins, the cytoskeletal protein talin has been shown to play a pivotal role in integrin-mediated events.

In a collaboration with David Critchley and with Igor Barsukov (Liverpool), involving an integrated combination of NMR, crystallography and biochemical & cell biological assays, we have been focussing on an analysis of the structure-function relationships in talin, a large (2541 residue) protein which consists of a globular N-terminal head (~50kDa), which contains a FERM domain, and a large flexible C-terminal rod (~220kDa) comprised of 62 helices that are organized into a series of helical bundles, followed by a single C-terminal helix that forms an antiparallel homodimer. We have determined the structures of all 18 domains (and some multiple domains) of talin by a combination of NMR, X-ray crystallography and SAXS. The talin head is an atypical FERM domain with an additional N-terminal sub-domain and with the sub-domains F0-F3 arranged in a linear fashion, appropriate for interaction with the membrane. We have defined the structure of a complex between the F3 domain of the talin head and a helical bundle domain of the rod, an intramolecular interaction which maintains ‘resting’ talin in a state where it cannot bind integrins. We have identified the binding sites on the rod for actin, RIAM and vinculin. Importantly, we have shown that the binding of vinculin and RIAM to the N-terminal region of the talin rod is competitive but occurs by different mechanisms. Vinculin binding involves unfolding of the helical bundles and is activated by mechanical force whereas RIAM binds to the folded bundles. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on the talin rod disrupts RIAM binding and exposes the vinculin binding sites, which recruit vinculin to stabilize the complex.

Selected recent publications on talin:

  • Goult, B.T., Bate, N., Anthis, N.J., Wegener, K.L., Gingras, A.R., Patel, B., Barsukov, I.L., Campbell, I.D., Roberts, G.C. K., and Critchley, D.R. (2009) The structure of an interdomain complex which regulates talin activity. J. Biol. Chem, 284, 15097-15106. doi:10.1074/jbc.M900078200. Selected as JBC Paper of the Week.
  • Gingras, A.R., Bate, N., Patel, B., Goult, B.T., Kopp, P.M., Emsley, J., Barsukov, I.L., Roberts, G.C.K. and Critchley, D.R. (2010) The central region of talin has a unique fold that binds vinculin and actin. J. Biol. Chem., 285, 29577–29587. doi:10.1074/jbc.M109.095455
  • Elliott, P.R., Goult, B.T., Kopp, P.M., Bate, N., Grossmann, J. G., Roberts, G. C. K., Critchley, D. R.,  and Barsukov, I. L. (2010) The structure of the talin head reveals a novel extended conformation of the FERM domain. Structure, 18, 1289–1299. doi:10.1016/j.str.2010.07.011
  • Goult, B.T., Zacharchenko, T., Bate, N., Gingras, A.R., Hey, F., Elliott, P.R., Roberts, G.C.K., Critchley, D.R., and Barsukov, I.L. (2013) RIAM and vinculin binding to talin are mutually exclusive and regulate adhesion assembly and turnover. J. Biol. Chem., 288, 8238-8249.  doi:10.1074/jbc.M112.438119. Recommended in F1000Prime

• NADPH-Cytochrome P450 Reductase and electron transfer to the drug-metabolising cytochromes P450.
The cytochrome P450 mono-oxygenase system in the endoplasmic reticulum plays a central role in drug metabolism and hence in the response of man to both therapeutic drugs and environmental toxins. Members of the cytochrome P450 superfamily share a common overall fold and catalytic mechanism, but differ markedly in their active site architecture, leading to very diverse substrate specificity. In man a group of only 6-7 P450s can account for the metabolism of 90-95% of drugs. We have used mutagenesis, modelling and NMR to study the structural basis of the specificity of two of the major human P450s, CYP2D6 and CYP3A4, as well as a biotechnologically important bacterial enzyme, CYP102A1. This has included the design of mutants with 800-fold increased affinity for substrate and mutants able to metabolise a classical inhibitor of P450 2D6.

P450s catalyse the insertion of one atom of molecular oxygen into their substrates with the reduction of the other atom to water, a reaction requiring two electrons. Biological electron transfer is generally carried out by proteins associated in large, dynamic complexes; often a single protein contains several redox centres, each in a structurally independent domain. This is indeed the case for drug-metabolising mono-oxygenases, in which electron transfer to the P450 is mediated by the multidomain flavoprotein NADPH-cytochrome P450 reductase (CPR). CPR accepts electrons from the obligatory two-electron donor NADPH onto its FAD cofactor and transfers them via its FMN cofactor to P450, the electrons being donated one at a time at two distinct steps in the reaction cycle of P450. The arrangement of CPR and the P450s in the membrane and the structure & dynamics of the complex(es) between them are clearly important for the electron transfer from CPR to P450, but consensus has not yet been reached on how they interact functionally. We have developed and characterised a reconstituted membrane system which allows measurement of the kinetics of electron transfer between the reductase and CYP3A4. Our kinetic data is consistent with a model in which the first electron transfer takes place within a relatively stable CPR–CYP3A4 complex but the complex can dissociate and re-form between the first and second electron transfers.

CPR comprises an N-terminal membrane anchor and three folded domains: a flavodoxin-like FMN-binding domain, an FAD- and NADPH-binding domain related to ferredoxin-NADP+reductase, and a ‘linker’ domain which may help to determine the mutual orientation of the domains. The FMN domain is connected to the FAD domain through a highly flexible ‘hinge’, and there is increasing evidence that domain movement is an essential part of the catalytic cycle of CPR. Our current work is focussed on the structural and functional characterisation of this domain movement, using small-angle X-ray and neutron scattering (SAXS and SANS), ion-mobility mass spectrometry, mutagenesis, NMR and kinetics. SAXS, SANS and ion-mobility MS experiments demonstrate that the reductase exists in a conformational equilibrium between a compact state appropriate for inter-flavin electron transfer and an extended state appropriate for electron transfer to P450s. Using the effects of changes in solution conditions and of site-directed mutagenesis, we have shown that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. Most significantly, our solution scattering results show unambiguously that a major domain reorganisation of this large enzyme is triggered by the delivery of electrons. It thus appears that the domain movement necessary for enzyme turnover is not achieved, as previously thought, by a random diffusive motion of the FMN domain in CPR. Rather, domain motion is linked closely to the individual electron delivery steps of the catalytic cycle of the enzyme, allowing a precise control of electron transfer in this complex system.

Current work, in collaboration with Professor Emma Raven (Chemistry), is focussed on:
(i)  developing and refining structural models for the extended conformation of CPR, using a combination of NMR, mutagenesis and SANS.
(ii)  using SANS to define the position of the conformational equilibrium in each intermediate in the catalytic cycle of CPR, under conditions where we can define the redox state unambiguously.
(iii)  determining the structure of the CPR-cytochrome complex.
(iv)  using neutron reflectometry to study the conformation of CPR and the formation of its complex with cytochrome P450 3A4 in lipid bilayer and nanodisc membranes.
Selected recent publications on CPR:

  • Ellis, J., Gutierrez, A., Barsukov, I. L., Huang, W.-C., Grossmann, J. G., and Roberts, G. C. K. (2009) Domain motion in cytochrome P450 reductase: conformational equilibria revealed by NMR and small-angle X-ray scattering. J. Biol. Chem., 284, 36628–36637 doi:10.1074/jbc.M109.054304. Selected as JBC Paper of the Week
  • Farooq, Y., and Roberts, G.C.K. (2010) Kinetics of electron transfer between cytochrome P450 reductase and cytochrome P450 3A4. Biochem. J., 432, 485-493. doi:10.1042/BJ20100744
  • Jenner, M., Ellis, J., Huang, W.-C., Raven, E.L., Roberts, G.C.K., and , N.J. (2011) Detection of a protein conformational equilibrium by electrospray ionisation-ion mobility-mass spectrometry. Angew. Chemie Int. Ed., 50, 8291-8294. doi: 10.1002/anie.201101077
  • Huang, W.-C., Ellis, J., Moody, P.C.E., Raven, E.L., and Roberts, G.C.K. (2013) Redox-linked domain movements in the catalytic cycle of cytochrome P450 reductase. Structure, 21, 1581-1589. doi: 10.1016/j.str.2013.06.022


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