Enhancing biochemical approaches to enable novel membrane protein structure determination by cryo-EM

MULTIPLE SCHOLARSHIPS ARE AVAILABLE 4-year Doctoral Program; Stipends: $33,413/year; Conference support: $5,000; Industry placement; Advanced training in all aspects of cryo-EM https://ccemmp.org/training/centre-doctoral-program/ OVERVIEW The Monash/MIPS node of the ARC Centre for Cryo-electron Microscopy of Membrane Proteins (CCeMMP) houses international leaders in the application of cryo-EM who are dedicated to the understanding of the structure and dynamics of G protein-coupled receptors (GPCRs), the largest family of cell surface receptors and a major target class for therapeutic drug development. There are over 800 GPCRs in the human genome including hundreds of receptors of potential therapeutic interest, including for treatment of metabolic, cardiovascular, inflammatory and CNS diseases. The program of research at MIPS has two arms, (i) technical advancement and (ii) application of cryo-EM to explore structure and function of GPCRs, and the application of this knowledge to drug discovery. MAJOR PROGRAM AREAS (i) Technology development In line with the advanced technical training that is a core feature of the Centre Doctoral Training Program, we are exploring ways to advance each of the 3 key areas in the workflow for cryo-EM of GPCRs from enabling biochemistry, to efficiency and robustness of vitrification and imaging, through to optimised data processing and the ability to directly derive 3D conformational dynamics. Enhancing biochemical approaches to expand the repertoire of GPCR states that can be studied by cryo-EM While robust methods have been developed for complexes of GPCRs and select G proteins (particularly Gs and Gi/o), optimisation of tools and/or conditions for routine complex formation with other G proteins and other transducers (eg. Arrestins) is required to fully exploit the potential of cryo-EM to understand agonist binding and receptor activation. Beyond this, there is a need to develop or optimise methods to adopt cryo-EM for study of other receptor states, including inactive/inhibitor bound receptors, apo receptors, receptor dimers and complexes of GPCRs with non-canonical G proteins (or other transducers) for structural interrogation of biased agonism. Solubilisation and membrane mimetic environments To date, almost all GPCR cryo-EM structures have been solved in detergent solubilised micelles. This project area will explore reconstitution into lipidic environments (eg. Using nanodiscs, SMALPs) and the influence of different lipid composition on formation and stability of GPCR complexes and the conformational dynamics of these complexes. Apo structures The instability of unliganded GPCRs has made determination of apo GPCR structure refractory to current approaches. Cryo-EM has the potential to address this gap in understanding of GPCR structure-function through innovation in sample preparation, imaging methods and data processing. Inactive structures While cryo-EM has become the method of choice for active-state, transducer-bound, GPCR complexes, inhibitor-bound, inactive structures have been the domain of x-ray crystallography. New tools and recent technical developments in imaging and analysis mean that cryo-EM is set to revolutionise the approach to inhibitor-bound structures. Higher order oligomers and application to drug discovery With the exception of recent structures of obligate dimers of class C GPCRs, the structural work to date has been limited to monomeric GPCR complexes. Structures of GPCR homo and hetero dimers could open up new avenues for novel drug development. Structural basis for biased agonism Biased agonism describes the ability of different ligands acting at the same receptor to differentially promote the recruitment of transducer proteins. To date, success in generation of stable complexes for structural imaging has been almost exclusively limited to the best coupled (canonical) G protein partner. However, to properly understand why one drug can favour a distinct pattern of transducer recruitment relative to another, structure of ligand-receptor complexes bound to different transducers is required. This poses questions on how to generate stable complexes for less well coupled G proteins/arrestins. Success in this project area has the potential to enable design of new drugs with predictable patterns of biased agonism. Our industry partners in the Centre currently include, Thermo Fisher Scientific, Astex, Servier, Pfizer, AstraZeneca, Novo Nordisk, Sanofi-Aventis and Dimerix Biosciences.