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

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基本情報

登録情報
データベース: PDB / ID: 2lv6
タイトルThe complex between Ca-Calmodulin and skeletal muscle myosin light chain kinase from combination of NMR and aqueous and contrast-matched SAXS data
要素
  • Calmodulin
  • Myosin light chain kinase 2, skeletal/cardiac muscle
キーワードMETAL BINDING PROTEIN/TRANSFERASE / Pb-substituted / protein complex / METAL BINDING PROTEIN-TRANSFERASE complex
機能・相同性
機能・相同性情報


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 / Calmodulin induced events / regulation of synaptic vesicle endocytosis / Reduction of cytosolic Ca++ levels / calcium/calmodulin-dependent protein kinase activity / 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 / 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 / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / regulation of cardiac muscle cell action potential / Activation of RAC1 downstream of NMDARs / response to corticosterone / positive regulation of DNA binding / positive regulation of ryanodine-sensitive calcium-release channel activity / nitric-oxide synthase binding / 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 / negative regulation of ryanodine-sensitive calcium-release channel activity / protein phosphatase activator activity / RHO GTPases activate PAKs / : / Ion transport by P-type ATPases / Long-term potentiation / Uptake and function of anthrax toxins / Regulation of MECP2 expression and activity / Calcineurin activates NFAT / adenylate cyclase binding / catalytic complex / DARPP-32 events / detection of calcium ion / regulation of cardiac muscle contraction / regulation of ryanodine-sensitive calcium-release channel activity / Smooth Muscle Contraction / cellular response to interferon-beta / RHO GTPases activate IQGAPs / calcium channel inhibitor activity / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / Protein methylation / phosphatidylinositol 3-kinase binding / eNOS activation / enzyme regulator activity / striated muscle contraction / activation of adenylate cyclase activity / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / Activation of AMPK downstream of NMDARs / cardiac muscle contraction / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / : / Ion homeostasis / titin binding / regulation of calcium-mediated signaling / positive regulation of protein autophosphorylation / voltage-gated potassium channel complex / sperm midpiece / calcium channel complex / response to amphetamine / 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 / FCERI mediated Ca+2 mobilization / protein serine/threonine kinase activator activity / FCGR3A-mediated IL10 synthesis / VEGFR2 mediated vascular permeability / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / VEGFR2 mediated cell proliferation / regulation of cytokinesis / positive regulation of peptidyl-threonine phosphorylation / positive regulation of nitric-oxide synthase activity / Translocation of SLC2A4 (GLUT4) to the plasma membrane
類似検索 - 分子機能
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 ...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
類似検索 - ドメイン・相同性
Calmodulin-1 / Calmodulin-3 / Myosin light chain kinase 2, skeletal/cardiac muscle
類似検索 - 構成要素
生物種Homo sapiens (ヒト)
手法溶液散乱 / 溶液NMR / rigid-body optimization, simulated annealing
Model detailsfewest violations, model 1
データ登録者Grishaev, A.V. / Anthis, N.J. / Clore, G.M.
引用ジャーナル: J.Am.Chem.Soc. / 年: 2012
タイトル: Contrast-matched small-angle X-ray scattering from a heavy-atom-labeled protein in structure determination: application to a lead-substituted calmodulin-peptide complex.
著者: Grishaev, A. / Anthis, N.J. / Clore, G.M.
履歴
登録2012年6月29日登録サイト: BMRB / 処理サイト: RCSB
改定 1.02013年2月20日Provider: repository / タイプ: Initial release
改定 1.12013年3月27日Group: Other
改定 1.22024年5月1日Group: Data collection / Database references / Derived calculations
カテゴリ: 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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構造の表示

構造ビューア分子:
MolmilJmol/JSmol

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集合体

登録構造単位
A: Calmodulin
B: Myosin light chain kinase 2, skeletal/cardiac muscle
ヘテロ分子


分子量 (理論値)分子数
合計 (水以外)19,8116
ポリマ－19,6512
非ポリマー1604
水00
1


  • 登録構造と同一
  • 登録者・ソフトウェアが定義した集合体
タイプ名称対称操作数
identity operation1_555x,y,z1
Buried area1442.5 Å2
ΔGint-23.7 kcal/mol
Surface area2977 Å2
手法PISA
NMR アンサンブル
データ基準
コンフォーマー数 (登録 / 計算)1 / 1structures with the least restraint violations
代表モデルモデル #1fewest violations

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要素

#1: タンパク質 Calmodulin / CaM


分子量: 16678.324 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト)
遺伝子: CALM1, CALM, CAM, CAM1, CALM2, CAM2, CAMB, CALM3, CALML2, CAM3, CAMC, CAMIII
発現宿主: Escherichia coli (大腸菌) / 株 (発現宿主): BL21 / Variant (発現宿主): codon-plus (DE3) RIPL / 参照: UniProt: P62158, UniProt: P0DP23*PLUS
#2: タンパク質・ペプチド Myosin light chain kinase 2, skeletal/cardiac muscle / MLCK2


分子量: 2972.538 Da / 分子数: 1 / Fragment: Calmodulin-binding residues 566-591 / 由来タイプ: 合成 / 由来: (合成) Homo sapiens (ヒト) / 参照: UniProt: Q9H1R3, myosin-light-chain kinase
#3: 化合物
ChemComp-CA / CALCIUM ION / カルシウムジカチオン


分子量: 40.078 Da / 分子数: 4 / 由来タイプ: 合成 / 式: Ca
配列の詳細THIS 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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実験情報

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実験

実験
手法詳細
溶液散乱This 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.
溶液NMR
NMR実験
Conditions-IDExperiment-IDSolution-IDタイプ
1112D 1H-15N HSQC
1222D 1H-15N HSQC

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試料調製

詳細
Solution-ID内容溶媒系
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
試料
濃度 (mg/ml)単位構成要素Isotopic 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
試料状態
Conditions-IDイオン強度pH圧 (kPa)温度 (K)
10.16.5ambient 300 K
20.156.5ambient 300 K

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データ収集

NMRスペクトロメータータイプ: Bruker DRX / 製造業者: Bruker / モデル: DRX / 磁場強度: 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 / 温度: 298 K / タイプ: x-ray

IDBuffer nameConc. range (mg/ml)Data analysis software listDetector specific検出器タイプMean 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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解析

NMR software
名称バージョン開発者分類
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Bax解析
SparkyGoddardデータ解析
CustomGrishaev構造決定
CNS1Brunger, Adams, Clore, Gros, Nilges and Read精密化
CustomGrishaev精密化
精密化手法: rigid-body optimization, simulated annealing / ソフトェア番号: 1
詳細: 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 ...詳細: 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.
代表構造選択基準: fewest violations
NMRアンサンブルコンフォーマー選択の基準: structures with the least restraint violations
計算したコンフォーマーの数: 1 / 登録したコンフォーマーの数: 1
Soln scatter modelコンフォーマー選択の基準: 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 ...コンフォーマー選択の基準: 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
詳細: 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 ...詳細: 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 / 代表コンフォーマー: 1 / Software list: PRIMUS, GNOM

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