Florey Department of Neuroscience and Mental Health - Theses
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ItemToward the structural characterisation of the relaxin receptor, RXFP1( 2021)Relaxin is a peptide hormone that is involved in several physiological processes such as pregnancy, collagen breakdown, fibrosis inhibition and vasodilation. It has been investigated for the use of several disease states such as scleroderma, fibrosis, cancer and most recently acute heart failure. Relaxin’s cognate receptor is the relaxin family peptide receptor 1 (RXFP1), an integral membrane protein belonging to the G protein-coupled receptor (GPCR) family with a complex, multistep activation mechanism which is still not well understood. Given the physiological roles of relaxin, RXFP1 is a promising target for the treatment of abovementioned conditions. However, there is currently a lack of a detailed mechanism in which relaxin mediated activation of RXFP1 occurs and this makes the design of relaxin-like compounds such as long active peptide mimetics, small molecules or biologics targeting RXFP1, or understanding and optimizing existing compounds that act at RXFP1 difficult. The lack of a detailed mechanism of RXFP1 activation can be attributed to the lack of full-length RXFP1 structures. While there are proposed models of this activation mechanism, these models were derived from studies on isolated domains of RXFP1 and thus it cannot be assumed that the findings are similar to that of a full-length RXFP1. Thus, the aim of this thesis was to work toward active and inactive state structures of full-length RXFP1 using cryo-electron microscopy (EM). By solving active and inactive state structures, we can overlay these structures to determine key conformational changes and key residues that interact with relaxin to determine a complete mode of relaxin mediated activation of RXFP1. However, these studies are hampered by the limitations of cryo-EM to study inactive state GPCRs and the low recombinant expression of WT RXFP1 which makes producing sufficient amounts of purified RXFP1 for these studies very difficult. In this thesis we optimised the expression and purification of RXFP1 for the purposes of cryo-EM studies. We also developed and optimised a novel tool, monomeric ultra-stable GFP (muGFP) as an intracellular loop 3 (ICL3) fusion partner to overcome the limitations of inactive state cryo-EM studies. We applied this to a thermostabilised variant of the alpha1A-adrenoceptor and demonstrated its utility for cryo-EM studies before applying it to RXFP1. Next, we applied an established workflow for the production of active state GPCR-G protein complexes in insect cells for cryo-EM studies to WT RXFP1 for the active state studies of the receptor. We also experimented with the expression and formation of an RXFP1-G protein complex in a mammalian expression systems. However, we were unable to proceed to cryo-EM studies of either inactive or active state RXFP1 due to inability to produce sufficient quantities of protein To overcome the limitation of poor protein yield, we developed a novel mammalian cell-based method of directed evolution. Existing methods of GPCR directed evolution are primarily E. coli based, and as RXFP1 is unable to be expressed in E. coli due to requiring post-translational modification, a mammalian system was required. We applied this novel method to RXFP1 and were able to evolve mutant #35, which demonstrated an ~9x increase in recombinant RXFP1 expression. Additionally, we also identified 2 mutants that demonstrated interesting pharmacological changes from WT. This includes a mutant that demonstrated an increase in basal signalling, and another mutant that demonstrates a decreased pEC50 for relaxin, that is a higher concentration of relaxin is required to produce an equivalent response in WT. By evolving high expressing mutant #35, we could potentially overcome the bottleneck of insufficient purified protein yield for cryo-EM studies. By applying mutant #35 to the workflows developed in this thesis, we can potentially enable downstream cryo-EM studies of RXFP1 through the ability to produce ~9x more protein than WT. Through enabling these studies, we may be able to elucidate the mechanism in which relaxin triggers RXFP1 activation in a full-length receptor. Understanding this mechanism in atomic resolution detail through cryo-EM studies could then facilitate rational drug design of novel relaxin-like mimetics for the treatment of acute heart failure or fibrosis or antagonists for the treatment of certain cancers.
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ItemProtein engineering of arginine vasopressin receptor V1A for structural biology( 2021)Peptide hormone arginine vasopressin (AVP) and its cognate G protein-coupled receptor (GPCR), V1A, belong to the vasopressin-oxytocin signalling system – a highly complex and widespread endocrine system in the human body. AVP activation of V1A causes a stimulatory contractile effect on vascular and uterine smooth muscle, while, in the brain, AVP and V1A play an important role in anxiety, aggression and social behaviours. Due to these physiological mechanisms, AVP and V1A have been of therapeutic interest for decades; initially for the treatment of peripheral conditions such as heart failure and menstrual cramps, while recently, V1A antagonists have shown promise for treating aspects of autism spectrum disorder (ASD). Despite this longstanding interest, there are currently no approved drugs selectively targeting V1A. Subtype-selectivity is important among the vasopressin-oxytocin receptor family, as these receptors share a high degree of sequence similarity and structural homology, yet have differing, and sometimes opposing, effects. Furthermore, as there are currently no experimental 3D structures of V1A, there is a lack of understanding on exactly how AVP binds to V1A at the molecular level, which limits the design and optimisation of new, V1A-selective drugs. GPCR structural biology is a rapidly developing field; however, the low-expression level and instability of many GPCRs, including V1A, can be problematic for purification and subsequent structural study. This thesis aimed to apply various protein engineering techniques to V1A, in order to facilitate the purification and subsequent biochemical study of this important, but long unfulfilled, potential therapeutic target. Chapter 2 covers the first GPCR-engineering venture, which was to introduce expression and stability augmenting missense mutations to V1A via a well-characterised method – directed evolution. This method, which uses E. coli display of receptor mutants, has successfully produced high-expressing, stabilised receptor mutants for the structural study of other neuropeptide receptors, including NTS1 and NK1. However, upon application of this method to V1A, stabilised V1A mutants were not produced. Instead, a V1A truncate was selected, which contained a small section of the V1A N terminus. The reason this protein was selected was unclear – but can primarily be attributed to the issues that arise when using E. coli for GPCR expression. It was evident that further engineering of V1A would require the use a different cell type, such as insect or mammalian cells. Chapter 3 explores the development of a mammalian cell-based directed evolution method. This new method was developed to overcome the expression issues encountered using E. coli, and required optimisation and innovation to accommodate the new cell type, including the use of lentivirus as the gene-delivery method. The method was developed, optimised and applied to V1A – producing V1A mutants that exhibited improved expression levels compared to wild-type V1A. Furthermore, several mutations were identified that appeared in multiple V1A mutants, indicating that they were contributing to this expression-boosting effect. Chapter 4 covers the purification of V1A using a combination of protein engineering approaches – including the introduction of the expression-boosting point mutations discovered in Chapter 3. Ultimately, functional V1A was able to be purified with the aid of these mutations, which provided a substantial increase in functional yield of purified receptor. The two main outcomes of this thesis are: the development of a mammalian cell-based directed evolution method that improves the functional yield of purified receptor – and the successful purification of functional V1A. Firstly, the new, mammalian cell-based selection method presents as a generic, rapid and viable means of improving the heterologous expression of low-expressing GPCRs – and potentially other complex membrane proteins with expression issues. Secondly, the substantial increase the yield of purified, functional V1A is a significant breakthrough in the biochemical study of V1A, as it enables the application of structural biology techniques, such as cryo-electron microscopy (cryo-EM), as well as ligand screening methods for new, V1A-selective ligands.