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- PDB-8if4: Structure of human alpha-2/delta-1 without mirogabalin -

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Basic information

Entry
Database: PDB / ID: 8if4
TitleStructure of human alpha-2/delta-1 without mirogabalin
ComponentsVoltage-dependent calcium channel subunit alpha-2/delta-1Voltage-gated calcium channel
KeywordsMEMBRANE PROTEIN / gabapentinoid / Cache domain / cryo-EM
Function / homology
Function and homology information


regulation of membrane repolarization during action potential / positive regulation of high voltage-gated calcium channel activity / 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 ...regulation of membrane repolarization during action potential / positive regulation of high voltage-gated calcium channel activity / 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 / neuronal dense core vesicle / regulation of heart rate by cardiac conduction / calcium ion import across plasma membrane / regulation of calcium ion transport / voltage-gated calcium channel activity / 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
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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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)125,7226
Polymers124,4131
Non-polymers1,3095
Water0
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 / 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.: 1
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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / 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
Has ligand of interestN

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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-1Voltage-gated calcium channel
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 FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 500 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: 331613 / Symmetry type: POINT
RefinementResolution: 3.2→116.28 Å / Cor.coef. Fo:Fc: 0.865 / ESU R: 0.567
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.43614 --
obs0.43614 74650 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 140.868 Å2
Refinement stepCycle: 1 / Total: 7424
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.0127589
ELECTRON MICROSCOPYr_bond_other_d0.0020.0166774
ELECTRON MICROSCOPYr_angle_refined_deg1.2181.64610302
ELECTRON MICROSCOPYr_angle_other_deg0.691.5615834
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.565907
ELECTRON MICROSCOPYr_dihedral_angle_2_deg4.9641038
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.063101282
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0530.21146
ELECTRON MICROSCOPYr_gen_planes_refined0.0020.028577
ELECTRON MICROSCOPYr_gen_planes_other0.0030.021486
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_it13.81413.8463652
ELECTRON MICROSCOPYr_mcbond_other13.79713.8473652
ELECTRON MICROSCOPYr_mcangle_it23.08420.724551
ELECTRON MICROSCOPYr_mcangle_other23.08220.7224552
ELECTRON MICROSCOPYr_scbond_it11.76715.0123937
ELECTRON MICROSCOPYr_scbond_other11.76515.0113938
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other20.55521.9855752
ELECTRON MICROSCOPYr_long_range_B_refined40.10730054
ELECTRON MICROSCOPYr_long_range_B_other40.10730055
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 -
Rwork1.302 5488 -
obs--100 %

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