{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/341805"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/341805","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Structure and activation of the class D GPCR dimer Ste2","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Velazhahan, Vaithish"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Tate, Christopher"],"committee_chairs":[],"committee_members":[],"year":2022,"date_issued":"2022-06-14","date_published":"2022-06-14","updated_at":"2026-07-22T22:24:17Z","subjects":["GPCRs","fungi","membrane proteins","cryo-EM","structure","G proteins","signalling","yeast"],"languages":["eng"],"rights":[],"rights_urls":["https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.89229","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Tate, Christopher"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Gates Cambridge Scholarship"]},{"key":"dc:creator","label":"Author","values":["Velazhahan, Vaithish"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2022-06-14"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/341805"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["GPCRs","fungi","membrane proteins","cryo-EM","structure","G proteins","signalling","yeast"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.89229"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1a1ad928-607c-43b7-a050-518382a8a654/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["55b084f88ee7ac84210b136a859d258b"]},{"key":"dc:title","label":"Title","values":["Structure and activation of the class D GPCR dimer Ste2"]}]}],"canonical_facts":{"dc:contributor.advisor":["Tate, Christopher"],"dc:contributor.sponsor":["Gates Cambridge Scholarship"],"dc:creator":["Velazhahan, Vaithish"],"dc:date.issued":["2022-06-14"],"dc:description.abstract":["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."],"dc:format.checksum.md5":["55b084f88ee7ac84210b136a859d258b"],"dc:identifier.doi":["10.17863/CAM.89229"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1a1ad928-607c-43b7-a050-518382a8a654/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/341805"],"dc:rights":["https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["GPCRs","fungi","membrane proteins","cryo-EM","structure","G proteins","signalling","yeast"],"dc:title":["Structure and activation of the class D GPCR dimer Ste2"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:17Z"}