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- PDB-8if3: Structure of human alpha-2/delta-1 with mirogabalin -

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Basic information

Entry
Database: PDB / ID: 8if3
TitleStructure of human alpha-2/delta-1 with mirogabalin
ComponentsVoltage-dependent calcium channel subunit alpha-2/delta-1
KeywordsMEMBRANE PROTEIN / gabapentinoid / Cache domain / cryo-EM
Function / homology
Function and homology information


regulation of membrane repolarization during action potential / calcium ion transmembrane transport via high voltage-gated calcium channel / membrane depolarization during bundle of His cell action potential / L-type voltage-gated calcium channel complex / cardiac muscle cell action potential involved in contraction / regulation of ventricular cardiac muscle cell membrane repolarization / calcium ion transport into cytosol / regulation of calcium ion transmembrane transport via high voltage-gated calcium channel / voltage-gated calcium channel complex / calcium ion import across plasma membrane ...regulation of membrane repolarization during action potential / calcium ion transmembrane transport via high voltage-gated calcium channel / membrane depolarization during bundle of His cell action potential / L-type voltage-gated calcium channel complex / cardiac muscle cell action potential involved in contraction / regulation of ventricular cardiac muscle cell membrane repolarization / calcium ion transport into cytosol / regulation of calcium ion transmembrane transport via high voltage-gated calcium channel / voltage-gated calcium channel complex / calcium ion import across plasma membrane / neuronal dense core vesicle / regulation of heart rate by cardiac conduction / regulation of calcium ion transport / voltage-gated calcium channel activity / presynaptic active zone membrane / GABA-ergic synapse / sarcoplasmic reticulum / cellular response to amyloid-beta / calcium ion transport / extracellular exosome / metal ion binding / plasma membrane
Similarity search - Function
VWA N-terminal / Voltage-dependent calcium channel, alpha-2/delta subunit, conserved region / VWA N-terminal / Neuronal voltage-dependent calcium channel alpha 2acd / : / von Willebrand factor type A domain / von Willebrand factor (vWF) type A domain / VWFA domain profile. / von Willebrand factor, type A / von Willebrand factor A-like domain superfamily
Similarity search - Domain/homology
Chem-8X9 / Voltage-dependent calcium channel subunit alpha-2/delta-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsKozai, D. / Numoto, N. / Fujiyoshi, Y.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H00451 Japan
Japan Agency for Medical Research and Development (AMED) Japan
CitationJournal: J Mol Biol / Year: 2023
Title: Recognition Mechanism of a Novel Gabapentinoid Drug, Mirogabalin, for Recombinant Human αδ1, a Voltage-Gated Calcium Channel Subunit.
Authors: Daisuke Kozai / Nobutaka Numoto / Kouki Nishikawa / Akiko Kamegawa / Shohei Kawasaki / Yoko Hiroaki / Katsumasa Irie / Atsunori Oshima / Hiroyuki Hanzawa / Kousei Shimada / Yutaka Kitano / Yoshinori Fujiyoshi /
Abstract: Mirogabalin is a novel gabapentinoid drug with a hydrophobic bicyclo substituent on the γ-aminobutyric acid moiety that targets the voltage-gated calcium channel subunit αδ1. Here, to reveal the ...Mirogabalin is a novel gabapentinoid drug with a hydrophobic bicyclo substituent on the γ-aminobutyric acid moiety that targets the voltage-gated calcium channel subunit αδ1. Here, to reveal the mirogabalin recognition mechanisms of αδ1, we present structures of recombinant human αδ1 with and without mirogabalin analyzed by cryo-electron microscopy. These structures show the binding of mirogabalin to the previously reported gabapentinoid binding site, which is the extracellular dCache_1 domain containing a conserved amino acid binding motif. A slight conformational change occurs around the residues positioned close to the hydrophobic group of mirogabalin. Mutagenesis binding assays identified that residues in the hydrophobic interaction region, in addition to several amino acid binding motif residues around the amino and carboxyl groups of mirogabalin, are critical for mirogabalin binding. The A215L mutation introduced to decrease the hydrophobic pocket volume predictably suppressed mirogabalin binding and promoted the binding of another ligand, L-Leu, with a smaller hydrophobic substituent than mirogabalin. Alterations of residues in the hydrophobic interaction region of αδ1 to those of the αδ2, αδ3, and αδ4 isoforms, of which αδ3 and αδ4 are gabapentin-insensitive, suppressed the binding of mirogabalin. These results support the importance of hydrophobic interactions in αδ1 ligand recognition.
History
DepositionFeb 17, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title
Revision 1.2Nov 20, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature / refine
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification ..._em_admin.last_update / _pdbx_entry_details.has_protein_modification / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Voltage-dependent calcium channel subunit alpha-2/delta-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,02011
Polymers124,4131
Non-polymers2,60710
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Voltage-dependent calcium channel subunit alpha-2/delta-1 / Voltage-gated calcium channel subunit alpha-2/delta-1


Mass: 124413.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CACNA2D1, CACNL2A, CCHL2A, MHS3 / Plasmid: BacMam / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: P54289
#2: 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.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#3: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#4: Chemical ChemComp-8X9 / 2-[(1R,5S,6S)-6-(aminomethyl)-3-ethyl-6-bicyclo[3.2.0]hept-3-enyl]acetic acid / Mirogabalin


Mass: 209.285 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C12H19NO2 / Feature type: SUBJECT OF INVESTIGATION
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: Voltage-dependent calcium channel subunit alpha-2/delta-1
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.14 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 68.3 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0352 / Classification: refinement
EM software
IDNameCategoryDetails
9Cootmodel refinement
10PHENIXmodel refinement
11CCP4 packagemodel refinementRefmac5 in Servalcat pipeline
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 207006 / Symmetry type: POINT
RefinementResolution: 3.23→3.23 Å / Cor.coef. Fo:Fc: 0.876 / ESU R: 0.542
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.40718 --
obs0.40718 73670 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 123.111 Å2
Refinement stepCycle: 1 / Total: 7528
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.0127699
ELECTRON MICROSCOPYr_bond_other_d0.0020.0166861
ELECTRON MICROSCOPYr_angle_refined_deg1.4061.65410460
ELECTRON MICROSCOPYr_angle_other_deg0.8171.56516045
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.9045909
ELECTRON MICROSCOPYr_dihedral_angle_2_deg4.1691039
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.189101288
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0640.21176
ELECTRON MICROSCOPYr_gen_planes_refined0.0030.028667
ELECTRON MICROSCOPYr_gen_planes_other0.0040.021497
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it14.06711.6023660
ELECTRON MICROSCOPYr_mcbond_other14.03911.6043660
ELECTRON MICROSCOPYr_mcangle_it22.96417.3474561
ELECTRON MICROSCOPYr_mcangle_other22.96317.3534562
ELECTRON MICROSCOPYr_scbond_it13.75413.464039
ELECTRON MICROSCOPYr_scbond_other13.75213.4654040
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other23.86419.5585898
ELECTRON MICROSCOPYr_long_range_B_refined41.82529663
ELECTRON MICROSCOPYr_long_range_B_other41.82629664
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.2→3.283 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.19 5441 -
obs--100 %

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