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Structure and activation of the class D GPCR dimer Ste2 10.17863/CAM.89229 Velazhahan, Vaithish Tate, Christopher G protein-coupled receptors (GPCRs) are eukaryotic membrane proteins that perform a broad range of cell signalling functions and are targets for approximately one-third of all FDA- approved drugs. GPCRs are divided phylogenetically into six classes, A-F. Over 700 structures of mammalian GPCRs (classes A, B, C and F) have been determined thus far, which greatly illuminate the structural mechanisms of GPCR-mediated signalling. However, structures were lacking for class D GPCRs, which are specific to fungi and are critical for their survival and reproduction. Ste2 (sterile-2 α-factor pheromone receptor) is a prototypical class D GPCR found in the baker’s yeast Saccharomyces cerevisiae and is critical for pheromone-sensing and sexual mating in yeast. Ste2 was the first ligand-binding GPCR to be sequenced, and advances in yeast genetics have allowed an extensive characterization of Ste2 and its pheromone-induced signalling pathway over the past three decades which has provided many paradigms for understanding the functions of GPCRs and G proteins. However, no structures of Ste2 were available to understand its molecular architecture and mechanistic basis of activation. This thesis describes five different structures of Ste2, including a ligand-free state, antagonist- bound state, two agonist-bound states, and an agonist-bound G protein-coupled state. These structures represent snapshots of Ste2 along its entire receptor activation pathway and reveal a new activation mechanism that is different from all other GPCRs studied thus far. The intracellular end of the helix H7 forms an irregular coil and sterically blocks the G protein coupling site in the inactive states. Upon agonist binding, there is a 6 Å outward movement of the extracellular end of H6, followed by a 20 Å outward movement of the intracellular end of H7 that unblocks the G protein coupling site. A 12 Å inward movement of the intracellular end of H6 also occurs so it can interact with the G protein. Ste2 exists as a homodimer in all the different states with an extensive dimer interface formed by the domain-swapped N-terminus, extracellular loop 1 and helix H1. The region immediately C-terminal of helix H7 in the inactive state transitions into an ordered α-helix upon Ste2 activation and contributes to the dimer interface only in the active states. The Ste2 dimer has evolved a fundamentally different mechanism to configure the movement of H6 and H7 upon agonist binding to allow G protein coupling and provides the first model for how interactions at the dimer interface can alter during receptor activation, which could have implications for understanding signalling in other transmembrane-mediated GPCR dimers. A mini-G protein was initially engineered to determine the active state G protein-coupled structure of Ste2. Two mini-G protein heterotrimers were found to couple simultaneously to agonist-bound Ste2. While one G protein heterotrimer was well-ordered, the other G protein was largely disordered, except for the C-terminal α5-helix of mini-Gpa1 that formed the majority of the contacts with the receptor. An additional structure of Ste2 coupled to two wild- type G protein heterotrimers was subsequently determined where both G proteins were ordered and this highlighted a potential inter-G protein interface between the two G protein heterotrimers. University of Cambridge 2022-06-14 Thesis Doctoral Doctor of Philosophy (PhD) eng Gates Cambridge Scholarship https://www.repository.cam.ac.uk/handle/1810/341805 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1a1ad928-607c-43b7-a050-518382a8a654/download 55b084f88ee7ac84210b136a859d258b https://www.rioxx.net/licenses/all-rights-reserved/ GPCRs fungi membrane proteins cryo-EM structure G proteins signalling yeast