Structure and function of membrane proteins important in synaptic signalling
<p>Membrane proteins are integral to synaptic signalling. This DPhil project explores the structures and functions of the muscle-type nicotinic acetylcholine receptor (AChR), a ligand-gated ion channel, and the SLC1 family of glutamate transporters.</p> <p>AChR is a pentameric lig...
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| Format: | Thesis |
| Language: | English |
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2024
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| _version_ | 1811139753879273472 |
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| author | Li, A |
| author2 | Dong, Y |
| author_facet | Dong, Y Li, A |
| author_sort | Li, A |
| collection | OXFORD |
| description | <p>Membrane proteins are integral to synaptic signalling. This DPhil project explores the structures and functions of the muscle-type nicotinic acetylcholine receptor (AChR), a ligand-gated ion channel, and the SLC1 family of glutamate transporters.</p>
<p>AChR is a pentameric ligand-gated ion channel that belongs to the same superfamily as GABAA, 5HT3, and glycine receptors. Despite the discovery of this protein more than a hundred years ago, much of its structural information is extrapolated from the <em>Torpedo</em> (electric ray) homolog. The human receptor includes a completely different subunit than <em>Torpedo</em> AChR, and its structure remained elusive due to the difficulty of expressing a hetero-pentameric protein with 4 subunits (α2βδε). I used lentivirus to generate a stable cell line expressing human AChR subunits. I then purified the protein by ligand-based affinity chromatography and solved its detergent-solubilised, α-bungarotoxin-bound, resting state structure to 3.1 Å. Further datasets of nanodisc-reconstituted AChR with α-bungarotoxin, or acetylcholine, or a mixture of activator and open pore blocker were collected, with ongoing data processing to capture the receptor in resting, desensitised, and open states. I demonstrated for the first time that human muscle-type AChR could be recombinantly expressed and used this expression method to elucidate the first structure of human muscle-type AChR.</p>
<p>SLC1A1 and SLC1A2 are glutamate transporters crucial for nutrient uptake and the removal of extracellular glutamate in the brain, thus helping to prevent excitotoxicity. Enhancing the transport activity of SLC1A2 can potentially limit the extent of neuronal death in neurodegenerative diseases and ischemic stroke. I solved the cryo-EM structure of the human full-length SLC1A2, the main glutamate reuptake transporter in the brain, to 3.2 Å. Whilst the activator compound GT949 was added to the sample, the molecule could not be identified from the cryo-EM map and thermal shift assay failed to support its interaction with purified SLC1A2 protein. Consequently, GT949 may not be a direct modulator of SLC1A2.</p>
<p>Furthermore, I screened 28 SLC1A1-directed camelid nanobodies by pulldown assay yielding 6 positive binders. The cryo-EM structure of one SLC1A1-nanobody complex was solved to 3.5 Å, showing a binding site on the intracellular side of the scaffold domain. Analysis of the binding site predicted 3 other possible binders to the same epitope and possible cross-reactivity to other SLC1s. This was the first nanobody epitope mapped onto SLC1s.</p>
<p>Together, these results provide important structural information on synaptic proteins as well as valuable tools and methods for future research and drug discovery.</p> |
| first_indexed | 2024-09-25T04:11:06Z |
| format | Thesis |
| id | oxford-uuid:c84db61e-504c-4066-9bfc-53be650f726d |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-09-25T04:11:06Z |
| publishDate | 2024 |
| record_format | dspace |
| spelling | oxford-uuid:c84db61e-504c-4066-9bfc-53be650f726d2024-06-20T11:50:32ZStructure and function of membrane proteins important in synaptic signallingThesishttp://purl.org/coar/resource_type/c_db06uuid:c84db61e-504c-4066-9bfc-53be650f726dProtein-protein interactionsBiochemistryIon channelsstructural biologyNeurosciencesMembrane proteinscryo-EMProtein complexesEnglishHyrax Deposit2024Li, ADong, YSauer, DBullock, APike, ANewstead, SMiller, P<p>Membrane proteins are integral to synaptic signalling. This DPhil project explores the structures and functions of the muscle-type nicotinic acetylcholine receptor (AChR), a ligand-gated ion channel, and the SLC1 family of glutamate transporters.</p> <p>AChR is a pentameric ligand-gated ion channel that belongs to the same superfamily as GABAA, 5HT3, and glycine receptors. Despite the discovery of this protein more than a hundred years ago, much of its structural information is extrapolated from the <em>Torpedo</em> (electric ray) homolog. The human receptor includes a completely different subunit than <em>Torpedo</em> AChR, and its structure remained elusive due to the difficulty of expressing a hetero-pentameric protein with 4 subunits (α2βδε). I used lentivirus to generate a stable cell line expressing human AChR subunits. I then purified the protein by ligand-based affinity chromatography and solved its detergent-solubilised, α-bungarotoxin-bound, resting state structure to 3.1 Å. Further datasets of nanodisc-reconstituted AChR with α-bungarotoxin, or acetylcholine, or a mixture of activator and open pore blocker were collected, with ongoing data processing to capture the receptor in resting, desensitised, and open states. I demonstrated for the first time that human muscle-type AChR could be recombinantly expressed and used this expression method to elucidate the first structure of human muscle-type AChR.</p> <p>SLC1A1 and SLC1A2 are glutamate transporters crucial for nutrient uptake and the removal of extracellular glutamate in the brain, thus helping to prevent excitotoxicity. Enhancing the transport activity of SLC1A2 can potentially limit the extent of neuronal death in neurodegenerative diseases and ischemic stroke. I solved the cryo-EM structure of the human full-length SLC1A2, the main glutamate reuptake transporter in the brain, to 3.2 Å. Whilst the activator compound GT949 was added to the sample, the molecule could not be identified from the cryo-EM map and thermal shift assay failed to support its interaction with purified SLC1A2 protein. Consequently, GT949 may not be a direct modulator of SLC1A2.</p> <p>Furthermore, I screened 28 SLC1A1-directed camelid nanobodies by pulldown assay yielding 6 positive binders. The cryo-EM structure of one SLC1A1-nanobody complex was solved to 3.5 Å, showing a binding site on the intracellular side of the scaffold domain. Analysis of the binding site predicted 3 other possible binders to the same epitope and possible cross-reactivity to other SLC1s. This was the first nanobody epitope mapped onto SLC1s.</p> <p>Together, these results provide important structural information on synaptic proteins as well as valuable tools and methods for future research and drug discovery.</p> |
| spellingShingle | Protein-protein interactions Biochemistry Ion channels structural biology Neurosciences Membrane proteins cryo-EM Protein complexes Li, A Structure and function of membrane proteins important in synaptic signalling |
| title | Structure and function of membrane proteins important in synaptic signalling |
| title_full | Structure and function of membrane proteins important in synaptic signalling |
| title_fullStr | Structure and function of membrane proteins important in synaptic signalling |
| title_full_unstemmed | Structure and function of membrane proteins important in synaptic signalling |
| title_short | Structure and function of membrane proteins important in synaptic signalling |
| title_sort | structure and function of membrane proteins important in synaptic signalling |
| topic | Protein-protein interactions Biochemistry Ion channels structural biology Neurosciences Membrane proteins cryo-EM Protein complexes |
| work_keys_str_mv | AT lia structureandfunctionofmembraneproteinsimportantinsynapticsignalling |