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- PDB-6w1j: Cryo-EM structure of 5HT3A receptor in presence of Alosetron -

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Basic information

Entry
Database: PDB / ID: 6w1j
TitleCryo-EM structure of 5HT3A receptor in presence of Alosetron
Components5-hydroxytryptamine receptor 3A
KeywordsTRANSPORT PROTEIN
Function / homology
Function and homology information


Neurotransmitter receptors and postsynaptic signal transmission / serotonin-gated monoatomic cation channel activity / serotonin-activated cation-selective channel complex / serotonin receptor signaling pathway / serotonin binding / inorganic cation transmembrane transport / excitatory extracellular ligand-gated monoatomic ion channel activity / cleavage furrow / transmembrane transporter complex / extracellular ligand-gated monoatomic ion channel activity ...Neurotransmitter receptors and postsynaptic signal transmission / serotonin-gated monoatomic cation channel activity / serotonin-activated cation-selective channel complex / serotonin receptor signaling pathway / serotonin binding / inorganic cation transmembrane transport / excitatory extracellular ligand-gated monoatomic ion channel activity / cleavage furrow / transmembrane transporter complex / extracellular ligand-gated monoatomic ion channel activity / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / transmembrane signaling receptor activity / presynaptic membrane / postsynaptic membrane / neuron projection / axon / neuronal cell body / glutamatergic synapse / synapse / identical protein binding / plasma membrane
Similarity search - Function
5-hydroxytryptamine 3 receptor / 5-hydroxytryptamine 3 receptor, A subunit / : / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor / Neurotransmitter-gated ion-channel ...5-hydroxytryptamine 3 receptor / 5-hydroxytryptamine 3 receptor, A subunit / : / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain
Similarity search - Domain/homology
alosetron / 5-hydroxytryptamine receptor 3A / 5-hydroxytryptamine (serotonin) receptor 3A
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsBasak, S. / Chakrapani, S.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM108921 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)3R01GM108921-03S1 United States
Citation
Journal: Elife / Year: 2020
Title: High-resolution structures of multiple 5-HTR-setron complexes reveal a novel mechanism of competitive inhibition.
Authors: Sandip Basak / Arvind Kumar / Steven Ramsey / Eric Gibbs / Abhijeet Kapoor / Marta Filizola / Sudha Chakrapani /
Abstract: Serotonin receptors (5-HTR) play a crucial role in regulating gut movement, and are the principal target of setrons, a class of high-affinity competitive antagonists, used in the management of nausea ...Serotonin receptors (5-HTR) play a crucial role in regulating gut movement, and are the principal target of setrons, a class of high-affinity competitive antagonists, used in the management of nausea and vomiting associated with radiation and chemotherapies. Structural insights into setron-binding poses and their inhibitory mechanisms are just beginning to emerge. Here, we present high-resolution cryo-EM structures of full-length 5-HTR in complex with palonosetron, ondansetron, and alosetron. Molecular dynamic simulations of these structures embedded in a fully-hydrated lipid environment assessed the stability of ligand-binding poses and drug-target interactions over time. Together with simulation results of apo- and serotonin-bound 5-HTR, the study reveals a distinct interaction fingerprint between the various setrons and binding-pocket residues that may underlie their diverse affinities. In addition, varying degrees of conformational change in the setron-5-HTR structures, throughout the channel and particularly along the channel activation pathway, suggests a novel mechanism of competitive inhibition.
#1: Journal: Nat Commun / Year: 2019
Title: Molecular mechanism of setron-mediated inhibition of full-length 5-HT receptor.
Authors: Sandip Basak / Yvonne Gicheru / Abhijeet Kapoor / Megan L Mayer / Marta Filizola / Sudha Chakrapani /
Abstract: Serotonin receptor (5-HTR) is the most common therapeutic target to manage the nausea and vomiting during cancer therapies and in the treatment of irritable bowel syndrome. Setrons, a class of ...Serotonin receptor (5-HTR) is the most common therapeutic target to manage the nausea and vomiting during cancer therapies and in the treatment of irritable bowel syndrome. Setrons, a class of competitive antagonists, cause functional inhibition of 5-HTR in the gastrointestinal tract and brainstem, acting as effective anti-emetic agents. Despite their prevalent use, the molecular mechanisms underlying setron binding and inhibition of 5-HTR are not fully understood. Here, we present the structure of granisetron-bound full-length 5-HTR solved by single-particle cryo-electron microscopy to 2.92 Å resolution. The reconstruction reveals the orientation of granisetron in the orthosteric site with unambiguous density for interacting sidechains. Molecular dynamics simulations and electrophysiology confirm the granisetron binding orientation and the residues central for ligand recognition. Comparison of granisetron-bound 5-HTR with the apo and serotonin-bound structures, reveals key insights into the mechanism underlying 5-HTR inhibition.
History
DepositionMar 4, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 13, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Assembly

Deposited unit
A: 5-hydroxytryptamine receptor 3A
B: 5-hydroxytryptamine receptor 3A
C: 5-hydroxytryptamine receptor 3A
D: 5-hydroxytryptamine receptor 3A
E: 5-hydroxytryptamine receptor 3A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)268,89526
Polymers260,2115
Non-polymers8,68421
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area39020 Å2
ΔGint-164 kcal/mol
Surface area97810 Å2

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Components

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Protein , 1 types, 5 molecules ABCDE

#1: Protein
5-hydroxytryptamine receptor 3A / 5-hydroxytryptamine receptor 3A short splice variant / Htr3a protein


Mass: 52042.121 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Htr3a / Cell line (production host): sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8K1F4, UniProt: P23979*PLUS

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Sugars , 3 types, 15 molecules

#2: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(4-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb4-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+4)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: Polysaccharide
beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#4: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE

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Non-polymers , 2 types, 6 molecules

#5: Chemical
ChemComp-S7Y / alosetron / 5-methyl-2-[(4-methyl-1~{H}-imidazol-5-yl)methyl]-3,4-dihydropyrido[4,3-b]indol-1-one


Mass: 294.351 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C17H18N4O / Feature type: SUBJECT OF INVESTIGATION / Comment: antagonist*YM
#6: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Serotonin receptor / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.27 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: sf9
Buffer solutionpH: 8
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 2.5 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 4350
Image scansMovie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
4Gctf1.06CTF correction
7Coot0.8.9model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.13-2998model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 568452
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42065 / Symmetry type: POINT
Atomic model buildingB value: 30 / Protocol: OTHER / Space: REAL

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