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- PDB-2lv6: The complex between Ca-Calmodulin and skeletal muscle myosin ligh... -

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Basic information

Entry
Database: PDB / ID: 2lv6
TitleThe complex between Ca-Calmodulin and skeletal muscle myosin light chain kinase from combination of NMR and aqueous and contrast-matched SAXS data
Components
  • Calmodulin
  • Myosin light chain kinase 2, skeletal/cardiac muscle
KeywordsMETAL BINDING PROTEIN/TRANSFERASE / Pb-substituted / protein complex / METAL BINDING PROTEIN-TRANSFERASE complex
Function / homology
Function and homology information


skeletal muscle satellite cell differentiation / regulation of muscle filament sliding / myosin-light-chain kinase / myosin light chain kinase activity / myosin light chain binding / neuromuscular synaptic transmission / : / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / cardiac muscle tissue morphogenesis ...skeletal muscle satellite cell differentiation / regulation of muscle filament sliding / myosin-light-chain kinase / myosin light chain kinase activity / myosin light chain binding / neuromuscular synaptic transmission / : / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / cardiac muscle tissue morphogenesis / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / regulation of synaptic vesicle endocytosis / Calmodulin induced events / Reduction of cytosolic Ca++ levels / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Activation of Ca-permeable Kainate Receptor / Loss of phosphorylation of MECP2 at T308 / CREB1 phosphorylation through the activation of Adenylate Cyclase / PKA activation / negative regulation of high voltage-gated calcium channel activity / calcium/calmodulin-dependent protein kinase activity / CaMK IV-mediated phosphorylation of CREB / Glycogen breakdown (glycogenolysis) / positive regulation of cyclic-nucleotide phosphodiesterase activity / organelle localization by membrane tethering / negative regulation of calcium ion export across plasma membrane / regulation of synaptic vesicle exocytosis / CLEC7A (Dectin-1) induces NFAT activation / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / Activation of RAC1 downstream of NMDARs / response to corticosterone / nitric-oxide synthase binding / positive regulation of ryanodine-sensitive calcium-release channel activity / regulation of cell communication by electrical coupling involved in cardiac conduction / Negative regulation of NMDA receptor-mediated neuronal transmission / negative regulation of peptidyl-threonine phosphorylation / Synthesis of IP3 and IP4 in the cytosol / Unblocking of NMDA receptors, glutamate binding and activation / Phase 0 - rapid depolarisation / skeletal muscle cell differentiation / protein phosphatase activator activity / RHO GTPases activate PAKs / positive regulation of phosphoprotein phosphatase activity / Ion transport by P-type ATPases / Long-term potentiation / Uptake and function of anthrax toxins / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / adenylate cyclase binding / catalytic complex / DARPP-32 events / detection of calcium ion / regulation of cardiac muscle contraction / negative regulation of ryanodine-sensitive calcium-release channel activity / Smooth Muscle Contraction / RHO GTPases activate IQGAPs / calcium channel inhibitor activity / cellular response to interferon-beta / positive regulation of DNA binding / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / Protein methylation / phosphatidylinositol 3-kinase binding / eNOS activation / striated muscle contraction / Activation of AMPK downstream of NMDARs / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / cardiac muscle contraction / enzyme regulator activity / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / positive regulation of protein dephosphorylation / Ion homeostasis / regulation of calcium-mediated signaling / regulation of ryanodine-sensitive calcium-release channel activity / titin binding / positive regulation of protein autophosphorylation / voltage-gated potassium channel complex / sperm midpiece / calcium channel complex / response to amphetamine / activation of adenylate cyclase activity / substantia nigra development / adenylate cyclase activator activity / Ras activation upon Ca2+ influx through NMDA receptor / nitric-oxide synthase regulator activity / regulation of heart rate / sarcomere / protein serine/threonine kinase activator activity / FCERI mediated Ca+2 mobilization / FCGR3A-mediated IL10 synthesis / VEGFR2 mediated vascular permeability / positive regulation of peptidyl-threonine phosphorylation / regulation of cytokinesis / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / VEGFR2 mediated cell proliferation / positive regulation of nitric-oxide synthase activity / Translocation of SLC2A4 (GLUT4) to the plasma membrane
Similarity search - Function
Myosin light chain kinase 2, catalytic domain / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. ...Myosin light chain kinase 2, catalytic domain / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Calmodulin-1 / Calmodulin-3 / Myosin light chain kinase 2, skeletal/cardiac muscle
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION SCATTERING / SOLUTION NMR / rigid-body optimization, simulated annealing
Model detailsfewest violations, model 1
AuthorsGrishaev, A.V. / Anthis, N.J. / Clore, G.M.
CitationJournal: J.Am.Chem.Soc. / Year: 2012
Title: Contrast-matched small-angle X-ray scattering from a heavy-atom-labeled protein in structure determination: application to a lead-substituted calmodulin-peptide complex.
Authors: Grishaev, A. / Anthis, N.J. / Clore, G.M.
History
DepositionJun 29, 2012Deposition site: BMRB / Processing site: RCSB
Revision 1.0Feb 20, 2013Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2013Group: Other
Revision 1.2May 1, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_nmr_software / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_nmr_software.name / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Calmodulin
B: Myosin light chain kinase 2, skeletal/cardiac muscle
hetero molecules


Theoretical massNumber of molelcules
Total (without water)19,8116
Polymers19,6512
Non-polymers1604
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1442.5 Å2
ΔGint-23.7 kcal/mol
Surface area2977 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)1 / 1structures with the least restraint violations
RepresentativeModel #1fewest violations

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Components

#1: Protein Calmodulin / CaM


Mass: 16678.324 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: CALM1, CALM, CAM, CAM1, CALM2, CAM2, CAMB, CALM3, CALML2, CAM3, CAMC, CAMIII
Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / Variant (production host): codon-plus (DE3) RIPL / References: UniProt: P62158, UniProt: P0DP23*PLUS
#2: Protein/peptide Myosin light chain kinase 2, skeletal/cardiac muscle / MLCK2


Mass: 2972.538 Da / Num. of mol.: 1 / Fragment: Calmodulin-binding residues 566-591 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: Q9H1R3, myosin-light-chain kinase
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
Sequence detailsTHIS DEPOSITED STRUCTURE OF HUMAN CALMODULIN WAS REFINED USING INDIVIDUAL DOMAIN COORDINATES OF ...THIS DEPOSITED STRUCTURE OF HUMAN CALMODULIN WAS REFINED USING INDIVIDUAL DOMAIN COORDINATES OF DEPOSITION 1MXE, WHICH CORRESPONDS TO THE CHICKEN CALMODULIN. THE SEQUENCES OF HUMAN AND CHICKEN CALMODULIN DIFFER IN TWO POSITIONS, 99 (TYR FOR HUMAN AND PHE FOR CHICKEN) AND 143 (GLN FOR HUMAN AND THR FOR CHICKEN). THEREFORE, THE SIDECHAIN COORDINATES OF RESIDUES 99 AND 143 IN THIS DEPOSITION DO NOT REPRESENT COMPOSITION OF THE SAMPLES USED FOR EXPERIMENTAL DATA COLLECTION. THIS DISCREPANCIES DO NOT PRODUCE ANY DIFFERENCES WHEN EXPERIMENTAL DATA ARE FITTED SINCE BACKBONE N-HN RDC AND CONTRAST-MATCHED SAXS DATA DO NOT INVOLVE THE SIDECHAIN COORDINATES AND THEIR IMPACT ON THE AQUEOUS SAXS DATA IS NEGLIGIBLE.

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Experimental details

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Experiment

Experiment
MethodDetails
SOLUTION SCATTERINGThis entry contains the coordinates of the complex between Ca-bound human calmodulin and skeletal muscle myosin light chain kinase determined by refinement against backbone HN-N residual dipolar couplings from solution NMR and solution X-ray scattering intensity data. The latter include both SAXS data in H2O recorded on a Ca-CAM/MLCK sample and data in 65% aqueous sucrose buffer recorded on the Pb-CaM/MLCK sample in which Ca ions in calmodulin were replaced by Pb ions. The position of the C-terminal relative to the N-terminal domain of calmodulin was optimized via an exhaustive rigid-body translational/orientational grid search with a step size of 1A/2deg while fitting RDCs and the two types of SAXS data. Subsequently, the coordinates of the linker connecting the two domain of calmodulin (residues 76-81) and the interfacial side chains were optimized via molecular dynamics/simulated annealing refinement while keeping the coordinates of the backbone atoms fixed.
SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 1H-15N HSQC
1222D 1H-15N HSQC

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Sample preparation

Details
Solution-IDContentsSolvent system
10.3 mM [U-13C; U-15N; U-2H] Calmodulin, 0.3 mM ssMLCK, 95% H2O/5% D2O95% H2O/5% D2O
20.06-0.25 mM [U-13C; U-15N; U-2H] Calmodulin, 0.06-0.25 mM ssMLCK, 65% w/v sucrose/H2O65% w/v sucrose/H2O
Sample
Conc. (mg/ml)UnitsComponentIsotopic labelingConc. range (mg/ml)Solution-ID
0.3 mMCalmodulin-1[U-13C; U-15N; U-2H]1
0.3 mMssMLCK-21
mMCalmodulin-3[U-13C; U-15N; U-2H]0.06-0.252
mMssMLCK-40.06-0.252
Sample conditions
Conditions-IDIonic strengthpHPressure (kPa)Temperature (K)
10.1 6.5 ambient 300 K
20.15 6.5 ambient 300 K

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Data collection

NMR spectrometerType: Bruker DRX / Manufacturer: Bruker / Model: DRX / Field strength: 600 MHz
Soln scatter

Data reduction software list: MARDETECTOR, IGOR / Num. of time frames: 20 / Sample pH: 6.5 / Source class: Y / Source type: ADVANCED PHOTON SOURCE / Temperature: 298 K / Type: x-ray

IDBuffer nameConc. range (mg/ml)Data analysis software listDetector specificDetector typeMean guiner radius (nm)Mean guiner radius esd (nm)Protein lengthSource beamline
125MM HEPES, 150 MM KCL, 3 MM CACL2, 1 MM TCEP, H2O1.2-5.0PRIMUS, GNOMDECTRISPILATUS 2M1.780.04612-IDB
225MM HEPES, 150 MM KCL, 1 MM TCEP, 3 MICROMOL PBCL2, 65% SUCROSE, H2O5.0 - 9.5PRIMUSGOLD CCD1.80.212-IDC

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Processing

NMR software
NameVersionDeveloperClassification
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
SparkyGoddarddata analysis
CustomGrishaevstructure solution
CNS1Brunger, Adams, Clore, Gros, Nilges and Readrefinement
CustomGrishaevrefinement
RefinementMethod: rigid-body optimization, simulated annealing / Software ordinal: 1
Details: Relative positions of the N- and C-terminal domains of calmodulin, rigidly held at those of the 1MXE structure, chain A, were optimized via a rotational/translational grid search with steps ...Details: Relative positions of the N- and C-terminal domains of calmodulin, rigidly held at those of the 1MXE structure, chain A, were optimized via a rotational/translational grid search with steps of 2deg and 1A, respectively with the best model fitted against RDC and scattering intensity data. Coordinates of the calmodulin linker residues (76-81) and the interfacial side chains were regularized via a molecular dynamics/simulated annealing protocol with the backbone atoms and the remaining side chains held fixed at the geometry resulting from the rigid-body grid search of the previous step.
NMR representativeSelection criteria: fewest violations
NMR ensembleConformer selection criteria: structures with the least restraint violations
Conformers calculated total number: 1 / Conformers submitted total number: 1
Soln scatter modelConformer selection criteria: BEST-FITTING CONFORMER BASED ON THE FIT OF AQUEOUS SAXS DATA FOR CA-CAM/MLCK, CONTRAST-MATCHED (65% SUCROSE) SAXS DATA FOR PB-CAM/MLCK, AND BACKBONE 1H-15N RESIDUAL ...Conformer selection criteria: BEST-FITTING CONFORMER BASED ON THE FIT OF AQUEOUS SAXS DATA FOR CA-CAM/MLCK, CONTRAST-MATCHED (65% SUCROSE) SAXS DATA FOR PB-CAM/MLCK, AND BACKBONE 1H-15N RESIDUAL DIPOLAR COUPLINGS FOR CAM IN THE CA-CAM/MLCK COMPLEX
Details: 4000000000000 STARTING RELATIVE POSITIONS OF THE N-TERM (1-75) AND C-TERM (82-148) DOMAINS OF CAM WERE BUILT USING COORDINATES IN THE PDB DEPOSITION 1MXE:A. TAIL RESIDUES 1-4 AND 144-148 ...Details: 4000000000000 STARTING RELATIVE POSITIONS OF THE N-TERM (1-75) AND C-TERM (82-148) DOMAINS OF CAM WERE BUILT USING COORDINATES IN THE PDB DEPOSITION 1MXE:A. TAIL RESIDUES 1-4 AND 144-148 WERE BEST-FITTED TO 1MXE USING MODELS 1J7O AND 1J7P. THESE 4*1011 MODELS SAMPLE IN AN EXHAUSTIVE FASHION ALL POSSIBLE RELATIVE DOMAIN POSITIONS WHICH DIFFER BY NO MORE THAN 1A IN TERMS OF C-TERM DOMAIN PLACEMENT WHEN N-TERM DOMAIN IS FIXED. ROTATIONAL AND TRANSLATIONAL GRIDS OF 2DEG AND 1A WERE USED. THE MODELS WERE THEN FILTERED TO EXHIBIT FIT TO THE RDC DATA NO WORSE THAN 10% HIGHER THAN GLOBAL MINIMUM FOUND BY NLS OPTIMIZATION OF ORIENTATION OF THE C-TERM RELATIVE TO THE N-TERM DOMAIN. ADDITIONAL FILTERS WERE THEN APPLIED INCLUDING ABSENCE OF CLASHES <2.5 A BETWEEN HEAVY BACKBONE AND CB ATOMS, AND RGYR OF 13-19A. FOR THE GEOMETRIES THAT PASSED ABOVE REQUIREMENTS, POSITION OF MLCK FROM 2BBM DEPOSITION WAS BEST-FITTED IN A RIGID-BODY MANNER AGAINST NOE DISTANCE RESTRAINTS FROM 2BBM DEPOSITION AND CLASH AVOIDANCE FUNCTION INCLUDING HEAVY BACKBONE AND CB ATOMS. CAM CONFORMATIONS WHICH EXHIBITED AT LEAST 1 NOE VIOLATION EXCEEDING 3A FOR THE BEST-FITTED MLCK WERE REJECTED. LINKER COORDINATES WERE THEN BUILT FOR RESIDUES 76-81 USING 8-RESIDUE BACKBONE AND CB-CONTAINING SEGMENTS FROM A PDB-BASED DATABASE INCLUDING 2.2 MILLION RESIDUES IN TOTAL. LINKERS WHICH CLASHED WITH CAM DOMAINS OR MLCK OR EXHIBITED RMSD ABOVE 1.2 A FOR RESIDUES 1 AND 8 VS RESIDUES 75 AND 82 OF CAM WERE REJECTED. CAM CONFORMATION FOR WHICH SUCH LINKERS COULD NOT BE BUILT WERE REJECTED LEADING TO A TOTAL OF APPROXIMATELY 75000 STRUCTURES. THESE STRUCTURES WERE FITTED TO THE SAXS AND RDC DATA WITH THE OVERALL BEST-FITTING MODEL RETAINED FOR DEPOSITION. THE COORDINATES OF THE LINKER RESIDUES 76-81 AND SIDECHAINS WERE REGULARIZED BY ENERGY MINIMIZATION VIA XPLOR-NIH PRIOR TO THE DEPOSITION WITH THE ACTIVE ENERGY TERMS INCLUDING BONDS, ANGLES, IMPROPERS, NON-BONDED, AND CONFORMATIONAL DATABASE POTENTIALS OF MEAN FORCE.
Num. of conformers submitted: 1 / Representative conformer: 1 / Software list: PRIMUS, GNOM

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