Structural studies of the human GABAB receptor using cryo-electron microscopy
<p>The metabotropic gamma-aminobutyric acid type-B receptor (GABABR) mediates slow and prolonged inhibitory neurotransmission in the mammalian nervous system. GABABR belongs to the Class C GPCR family and is the first GPCR reported to function as an obligate heterodimer. Structurally, GABABR d...
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
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2018
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| _version_ | 1797107547913584640 |
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| author | Sun, Z |
| author2 | Aricescu, R |
| author_facet | Aricescu, R Sun, Z |
| author_sort | Sun, Z |
| collection | OXFORD |
| description | <p>The metabotropic gamma-aminobutyric acid type-B receptor (GABABR) mediates slow and prolonged inhibitory neurotransmission in the mammalian nervous system. GABABR belongs to the Class C GPCR family and is the first GPCR reported to function as an obligate heterodimer. Structurally, GABABR displays distinctive features for its lack of the cysteine-rich domain found conserved among other Class C receptors. GABABR’s extracellular Venus flytrap (VFT) domain is directly connected to the transmembrane domain (TMD), followed by a small cytoplasmic domain. To date, structures of an isolated extracellular VFT domain and an intracellular coiled-coil domain have been solved using X-ray crystallography. No structure of the transmembrane domain (TMD) or the full-length receptor has been reported.</p>
<p>This project focused on the recombinant production and structural analysis of the full-length human GABABR using single-particle cryo-Electron Microcopy (cryo-EM). It was determined that the HEK293 GnTI- cells were a suitable host cell system for production of the integral human GABABR. Following extensive optimisation of constructs and expression conditions, stable cell lines were generated which yielded sufficient material for structural work. GABABR was isolated from the membranes using a combination of detergents that maintained the receptor’s ligand binding activity. The high purity heterodimeric GABABR samples obtained were used to screen various reconstitution systems compatible with cryo-EM, including amphipols, salipro particles and MSP nanodiscs. The MSP2N2 lipid nanodisc was found to be optimal for reconstituting GABABR, yielding intact receptor capable of binding to G protein. Thus far, medium resolution ( 10 ~ 15Å) models have been reconstructed for the full-length GABABR. These models provided new insights regarding GABABR domain organisations and receptor dynamics. In addition, a ~4Å reconstruction was obtained for the extracellular VFT domain alone and showed density for regions missing in the published crystal structures. The work presented in this thesis provides the foundation for solving higher-resolution GABABR structures and for their detailed mechanistic investigation.</p> |
| first_indexed | 2024-03-07T07:16:11Z |
| format | Thesis |
| id | oxford-uuid:03a89962-6b9e-401e-941c-16b24c4650d8 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:16:11Z |
| publishDate | 2018 |
| record_format | dspace |
| spelling | oxford-uuid:03a89962-6b9e-401e-941c-16b24c4650d82022-08-25T10:21:53ZStructural studies of the human GABAB receptor using cryo-electron microscopyThesishttp://purl.org/coar/resource_type/c_db06uuid:03a89962-6b9e-401e-941c-16b24c4650d8EnglishORA Deposit2018Sun, ZAricescu, RHuiskonen, J<p>The metabotropic gamma-aminobutyric acid type-B receptor (GABABR) mediates slow and prolonged inhibitory neurotransmission in the mammalian nervous system. GABABR belongs to the Class C GPCR family and is the first GPCR reported to function as an obligate heterodimer. Structurally, GABABR displays distinctive features for its lack of the cysteine-rich domain found conserved among other Class C receptors. GABABR’s extracellular Venus flytrap (VFT) domain is directly connected to the transmembrane domain (TMD), followed by a small cytoplasmic domain. To date, structures of an isolated extracellular VFT domain and an intracellular coiled-coil domain have been solved using X-ray crystallography. No structure of the transmembrane domain (TMD) or the full-length receptor has been reported.</p> <p>This project focused on the recombinant production and structural analysis of the full-length human GABABR using single-particle cryo-Electron Microcopy (cryo-EM). It was determined that the HEK293 GnTI- cells were a suitable host cell system for production of the integral human GABABR. Following extensive optimisation of constructs and expression conditions, stable cell lines were generated which yielded sufficient material for structural work. GABABR was isolated from the membranes using a combination of detergents that maintained the receptor’s ligand binding activity. The high purity heterodimeric GABABR samples obtained were used to screen various reconstitution systems compatible with cryo-EM, including amphipols, salipro particles and MSP nanodiscs. The MSP2N2 lipid nanodisc was found to be optimal for reconstituting GABABR, yielding intact receptor capable of binding to G protein. Thus far, medium resolution ( 10 ~ 15Å) models have been reconstructed for the full-length GABABR. These models provided new insights regarding GABABR domain organisations and receptor dynamics. In addition, a ~4Å reconstruction was obtained for the extracellular VFT domain alone and showed density for regions missing in the published crystal structures. The work presented in this thesis provides the foundation for solving higher-resolution GABABR structures and for their detailed mechanistic investigation.</p> |
| spellingShingle | Sun, Z Structural studies of the human GABAB receptor using cryo-electron microscopy |
| title | Structural studies of the human GABAB receptor using cryo-electron microscopy |
| title_full | Structural studies of the human GABAB receptor using cryo-electron microscopy |
| title_fullStr | Structural studies of the human GABAB receptor using cryo-electron microscopy |
| title_full_unstemmed | Structural studies of the human GABAB receptor using cryo-electron microscopy |
| title_short | Structural studies of the human GABAB receptor using cryo-electron microscopy |
| title_sort | structural studies of the human gabab receptor using cryo electron microscopy |
| work_keys_str_mv | AT sunz structuralstudiesofthehumangababreceptorusingcryoelectronmicroscopy |