Integral membrane proteins are encoded by a large proportion of the genome of organisms across all kingdoms of life, account for more than half of the drug targets – and yet have traditionally been severely neglected by structural, functional and mechanistic studies. Our focus is thus on understanding these proteins in complete functional detail, starting from static structures and working towards a complete dynamical mechanism. We use such understanding as the basis for inhibitor and drug design. Our primary techniques include X-ray crystallography, cryo-electron microscopy transport and other functional assays. We also collaborate with other groups on FRET, PELDOR and electrometric measurements. We are thus dedicated to contributing as much as we can to the current and future study of these highly clinically, agriculturally and mechanistically relevant proteins.
Current major projects
- Integral membrane pyrophosphatases
- Transporters as drug targets
- Bacterial trimeric autotransporters and immune evasion
- Improving the membrane protein pipeline
Detailed research programme
Integral membrane pyrophosphatases
Membrane integral pyrophosphatases are clinically and agriculturally relevant enzymes that couple the break-down of pyrophosphate to the pumping of a cation across a membrane. We published the first high-resolution structure of a sodium-pumping enzyme in 2012. These are validated drug targets in protozoan parasites. We aim to gain in-depth structural and mechanistic understanding of by traditional and serial crystallography, enzymatic and cation pumping activity measurements, small molecule screening in vitro and in silico, in silico using Molecular Dynamics and in vitro using PELDOR and FRET. We collaborate with Andreas Kalli, Roman Tuma, Colin Fishwick, Christos Pliotas, Arwen Pearson (Hamburg) and Molecular Dimensions.
Transporters as drug targets
Transporters are potential targets for several diseases: as novel bacterial targets and/or to enhance antibiotic effectiveness, to enhance the effectiveness of anticancer drugs and as antiviral targets. We study a variety of efflux and amino-acid importers from Mycobacterium spp, with the focus on ones from M. tuberculosis, the causative agent of tuberculosis. Using large-scale screens, we have been able to purify a number of them to homogeneity, have identified the molecules they transport and are studying them structurally and functionally. In a similar vein, with the Henderson group, we also study the novel family of Proteobacterial Antimicrobial Compound Efflux (PACE) transporters found only in Gram-negative bacteria and of unknown structure. Our vision is to develop novel inhibitors.
Bacterial trimeric autotransporters and immune evasion
Trimeric autotransporters (Type Vc secretion system) are some of the largest proteins found in Gram-negative bacteria, with sizes as large as 1 MDa for a trimer. They are highly modular, repetitive proteins (figure) that have evolved to provide both immune evasion by binding to key molecules and methods of binding to host cells and to extracellular matrix molecules. We are interested in understanding how they bind their ligands, for which there is as yet no structural information; what their their rigidity and affinity for ligands is under flow conditions and how they are transported through the outer membrane. Our vision is that this will lead to novel methods of combatting bacterial infections.
Improving the membrane protein pipeline
We are also heavily invested in improving the ease of membrane protein study. Only a fraction of the available solved structures are membrane proteins: they are hard to express and purify in a stable form. Our cutting edge approaches aim to reduce these issues. For instance, one of the most common contaminants following IMAC purification from E. coli is AcrB. By fractional factorial techniques, we have identified which His residues cause AcrB to copurify (figure of AcrB) and have developed a more “protein purification friendly” E. coli strain. Our novel pipeline IMPROvER to identify stabilising mutations has proved highly successful in four case studies and is now being applied to industrial targets. We collaborate with Steve Harborne (Peak Proteins).