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- PDB-6c0a: Actinin-1 EF-Hand bound to the Cav1.2 IQ Motif -

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Basic information

Entry
Database: PDB / ID: 6c0a
TitleActinin-1 EF-Hand bound to the Cav1.2 IQ Motif
Components
  • Alpha-actinin-1
  • Voltage-dependent L-type calcium channel subunit alpha-1C
KeywordsSIGNALING PROTEIN / actinin-1 / EF hand / Calcium Calmodulin / Neuronal signaling / Voltage Gated Calcium Channel / Cav1.2 / Voltage Gated Calcium Channel 1.2 / Hippocampal Neurons / IQ Motif
Function / homology
Function and homology information


cytoskeletal regulatory protein binding / dense core granule membrane / : / Regulation of cytoskeletal remodeling and cell spreading by IPP complex components / platelet morphogenesis / structural constituent of postsynapse / : / voltage-gated calcium channel activity involved in AV node cell action potential / voltage-gated calcium channel activity involved in cardiac muscle cell action potential / immune system development ...cytoskeletal regulatory protein binding / dense core granule membrane / : / Regulation of cytoskeletal remodeling and cell spreading by IPP complex components / platelet morphogenesis / structural constituent of postsynapse / : / voltage-gated calcium channel activity involved in AV node cell action potential / voltage-gated calcium channel activity involved in cardiac muscle cell action potential / immune system development / actin filament network formation / membrane depolarization during atrial cardiac muscle cell action potential / Phase 2 - plateau phase / calcium ion transmembrane transport via high voltage-gated calcium channel / membrane depolarization during AV node cell action potential / Platelet degranulation / vinculin binding / positive regulation of adenylate cyclase activity / cardiac conduction / L-type voltage-gated calcium channel complex / cortical cytoskeleton organization / fascia adherens / membrane depolarization during cardiac muscle cell action potential / regulation of ventricular cardiac muscle cell action potential / cardiac muscle cell action potential involved in contraction / high voltage-gated calcium channel activity / camera-type eye development / focal adhesion assembly / NCAM1 interactions / embryonic forelimb morphogenesis / calcium ion transport into cytosol / cell communication by electrical coupling involved in cardiac conduction / platelet formation / cortical cytoskeleton / voltage-gated calcium channel complex / regulation of heart rate by cardiac conduction / Phase 0 - rapid depolarisation / alpha-actinin binding / calcium ion import across plasma membrane / actin filament bundle assembly / brush border / voltage-gated calcium channel activity / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / stress fiber / ruffle / sarcomere / nuclear receptor coactivator activity / secretory granule / cell projection / Regulation of insulin secretion / actin filament organization / actin filament / calcium ion transmembrane transport / postsynaptic density membrane / Adrenaline,noradrenaline inhibits insulin secretion / Z disc / actin filament binding / cell-cell junction / double-stranded RNA binding / integrin binding / cell junction / heart development / positive regulation of cytosolic calcium ion concentration / perikaryon / transmembrane transporter binding / dendritic spine / postsynaptic density / cytoskeleton / calmodulin binding / protein domain specific binding / focal adhesion / dendrite / glutamatergic synapse / calcium ion binding / protein homodimerization activity / membrane / metal ion binding / nucleus / plasma membrane / cytoplasm
Similarity search - Function
Voltage-dependent calcium channel, L-type, alpha-1C subunit / Voltage-gated calcium channel subunit alpha, C-terminal / Voltage-gated calcium channel subunit alpha, C-term / Voltage-dependent calcium channel, L-type, alpha-1 subunit / EF-hand, Ca insensitive / Ca2+ insensitive EF hand / Voltage-dependent calcium channel, alpha-1 subunit, IQ domain / Voltage gated calcium channel IQ domain / Voltage gated calcium channel IQ domain / Voltage-dependent calcium channel, alpha-1 subunit ...Voltage-dependent calcium channel, L-type, alpha-1C subunit / Voltage-gated calcium channel subunit alpha, C-terminal / Voltage-gated calcium channel subunit alpha, C-term / Voltage-dependent calcium channel, L-type, alpha-1 subunit / EF-hand, Ca insensitive / Ca2+ insensitive EF hand / Voltage-dependent calcium channel, alpha-1 subunit, IQ domain / Voltage gated calcium channel IQ domain / Voltage gated calcium channel IQ domain / Voltage-dependent calcium channel, alpha-1 subunit / Voltage-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / Spectrin repeat / Spectrin repeat / Actinin-type actin-binding domain signature 1. / Actinin-type actin-binding domain signature 2. / Actinin-type actin-binding domain, conserved site / Spectrin/alpha-actinin / Spectrin repeats / Calponin homology domain / Calponin homology (CH) domain / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile. / Voltage-dependent channel domain superfamily / EF-hand / Recoverin; domain 1 / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / Ion transport domain / Ion transport protein / EF-hand domain / EF-hand domain pair / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Voltage-dependent L-type calcium channel subunit alpha-1C / Alpha-actinin-1
Similarity search - Component
Biological speciesMus musculus (house mouse)
Homo sapiens (human)
MethodSOLUTION NMR / simulated annealing
AuthorsTurner, M.L. / Ames, J.B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)EY012347 United States
Citation
Journal: To be Published
Title: Structural Basis of the Localization and Activation of Neuronal L-type Ca2+ Channels by a-Actinin1 and Calmodulin
Authors: Turner, M.L. / Anderson, D.E. / Coleman, A. / Horn, M. / Navedo, M. / Ames, J.B. / Hell, J.W.
#1: Journal: Biomol NMR Assign / Year: 2016
Title: Chemical shift assignments of the C-terminal EF-hand domain of a-actinin-1.
Authors: Turner, M. / Anderson, D.E. / Rajan, S. / Hell, J.W. / Ames, J.B.
History
DepositionDec 28, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 16, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 20, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Dec 11, 2019Group: Author supporting evidence / Data collection
Category: pdbx_audit_support / pdbx_nmr_software / pdbx_nmr_spectrometer
Item: _pdbx_audit_support.funding_organization / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model
Revision 1.3Jun 14, 2023Group: Database references / Other / Category: database_2 / pdbx_database_status
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Alpha-actinin-1
B: Voltage-dependent L-type calcium channel subunit alpha-1C


Theoretical massNumber of molelcules
Total (without water)10,7692
Polymers10,7692
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: isothermal titration calorimetry, Light Scatter was performed as well as Fluorescent polarization to confirm binding.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1420 Å2
ΔGint-11 kcal/mol
Surface area6310 Å2
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)4 / 194structures with the lowest energy
RepresentativeModel #1none

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Components

#1: Protein Alpha-actinin-1 / / Alpha-actinin cytoskeletal isoform / F-actin cross-linking protein / Non-muscle alpha-actinin-1


Mass: 7848.695 Da / Num. of mol.: 1 / Fragment: EF-hand 3,4 (UNP residues 822-892)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Actn1
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q7TPR4
#2: Protein/peptide Voltage-dependent L-type calcium channel subunit alpha-1C / Calcium channel / L type / alpha-1 polypeptide / isoform 1 / cardiac muscle / Voltage-gated calcium ...Calcium channel / L type / alpha-1 polypeptide / isoform 1 / cardiac muscle / Voltage-gated calcium channel subunit alpha Cav1.2


Mass: 2920.431 Da / Num. of mol.: 1 / Fragment: IQ Motif (UNP residues 1587-1609) / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: Q13936

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic12D 1H-15N HSQC
123isotropic13D 1H-15N NOESY
133isotropic13D HNCA
143isotropic13D HN(CA)CB
153isotropic13D CBCA(CO)NH
163isotropic23D (H)CCH-TOCSY
173isotropic13D CC(CO)NH
183isotropic2C13 NOESY
193isotropic2C13 CT HSQC
1104isotropic22D 1H-15N HSQC
1115isotropic22D 1H-15N HSQC

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution1300 uM [U-99% 15N] Actinin-1 EF hand, 300 uM IQ Motif, 90% H2O/10% D2O15N labeled Actinin-1 EF hand 3,4 with unlabeled IQ Motif15N90% H2O/10% D2O
solution2300 uM [U-99% 15N] Cav1.2 IQ Motif, 300 uM Actinin-1 EF 3,3, 90% H2O/10% D2O15N labeled Cav1.2 IQ Motif bound to unlabeled actinin-1 EF3,415N, unlabeled90% H2O/10% D2O
solution3300 uM [U-99% 13C; U-99% 15N] Actinin-1 EF 3,4, 300 uM Cav1.2 IQ Motif, 90% H2O/10% D2ODouble Labeled Actinin-1 EF 3,4 bound to IQ Motif15N, 13C90% H2O/10% D2O
gel solution412 mg/mL Pf1 phage, 300 uM [U-99% 15N] Actinin-1 EF 3,4, 300 uM Cav1.2 IQ Motif, 90% H2O/10% D2OSamples created with 12 mg/mL Pf1 phage for RDC calculation15N, Natural Abundance90% H2O/10% D2O
gel solution512 mg/mL Pf1 phage, 300 uM [U-99% 15N] Cav1.2 IQ Motif, 300 uM Actinin-1 EF 3,4, 90% H2O/10% D2OSamples created with 12 mg/mL Pf1 phage for RDC calculation15N, Natural Abundance_RDC90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
300 uMActinin-1 EF hand[U-99% 15N]1
300 uMIQ Motifnatural abundance1
300 uMCav1.2 IQ Motif[U-99% 15N]2
300 uMActinin-1 EF 3,3natural abundance2
300 uMActinin-1 EF 3,4[U-99% 13C; U-99% 15N]3
300 uMCav1.2 IQ Motifnatural abundance3
12 mg/mLPf1 phagenatural abundance4
300 uMActinin-1 EF 3,4[U-99% 15N]4
300 uMCav1.2 IQ Motifnatural abundance4
12 mg/mLPf1 phagenatural abundance5
300 uMCav1.2 IQ Motif[U-99% 15N]5
300 uMActinin-1 EF 3,4natural abundance5
Sample conditionsDetails: All samples were performed with the following buffer system: 20 mM Tris-d11 (pH 7.5) with 1 mM DTT d10, 1 mM EDTA-d12 and 95 % H2O/5 % D2O
Ionic strength: 0 mM / Label: Sample Conditions all Samples / pH: 7.5 / Pressure: 1 atm / Temperature: 303 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCEBrukerAVANCE8001
Bruker AVANCEBrukerAVANCE6002

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Processing

NMR software
NameDeveloperClassification
SparkyGoddardchemical shift assignment
TopSpinBruker Biospincollection
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
HADDOCKBonvinstructure calculation
PALESMarkus Zweckstetter and Ad Bax J. Am. Chem. Soc. , 122, (2000) 3791-3792 Contact: zweckste@speck.niddk.nih.govrefinement
SparkyGoddardpeak picking
RefinementMethod: simulated annealing / Software ordinal: 2 / Details: also distance geometry and torsion angle dynamics
NMR representativeSelection criteria: none
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 194 / Conformers submitted total number: 4

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