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- PDB-2kug: Halothane binds to druggable sites in calcium-calmodulin: Solutio... -

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Basic information

Entry
Database: PDB / ID: 2kug
TitleHalothane binds to druggable sites in calcium-calmodulin: Solution Structure of halothane-CaM N-terminal domain
ComponentsCalmodulin
KeywordsMETAL BINDING PROTEIN / CALMODULIN / CALCIUM BINDING / VOLATILE ANESTHETIC / HALOTHANE / Cytoskeleton / Isopeptide bond / Methylation / Phosphoprotein
Function / homology
Function and homology information


: / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / Reduction of cytosolic Ca++ levels / Activation of Ca-permeable Kainate Receptor / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde ...: / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / Reduction of cytosolic Ca++ levels / Activation of Ca-permeable Kainate Receptor / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / regulation of synaptic vesicle endocytosis / 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 / regulation of synaptic vesicle exocytosis / CaMK IV-mediated phosphorylation of CREB / Glycogen breakdown (glycogenolysis) / negative regulation of calcium ion export across plasma membrane / organelle localization by membrane tethering / Activation of RAC1 downstream of NMDARs / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / CLEC7A (Dectin-1) induces NFAT activation / autophagosome membrane docking / response to corticosterone / positive regulation of ryanodine-sensitive calcium-release channel activity / Negative regulation of NMDA receptor-mediated neuronal transmission / nitric-oxide synthase binding / regulation of cell communication by electrical coupling involved in cardiac conduction / Unblocking of NMDA receptors, glutamate binding and activation / negative regulation of peptidyl-threonine phosphorylation / Synthesis of IP3 and IP4 in the cytosol / Phase 0 - rapid depolarisation / protein phosphatase activator activity / RHO GTPases activate PAKs / positive regulation of cyclic-nucleotide phosphodiesterase activity / positive regulation of phosphoprotein phosphatase activity / Long-term potentiation / Ion transport by P-type ATPases / Uptake and function of anthrax toxins / adenylate cyclase binding / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / catalytic complex / DARPP-32 events / detection of calcium ion / Smooth Muscle Contraction / negative regulation of ryanodine-sensitive calcium-release channel activity / RHO GTPases activate IQGAPs / cellular response to interferon-beta / regulation of cardiac muscle contraction / calcium channel inhibitor activity / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / positive regulation of DNA binding / Protein methylation / enzyme regulator activity / voltage-gated potassium channel complex / phosphatidylinositol 3-kinase binding / Activation of AMPK downstream of NMDARs / eNOS activation / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / regulation of calcium-mediated signaling / positive regulation of protein dephosphorylation / titin binding / regulation of ryanodine-sensitive calcium-release channel activity / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Ion homeostasis / positive regulation of protein autophosphorylation / sperm midpiece / response to amphetamine / calcium channel complex / activation of adenylate cyclase activity / substantia nigra development / adenylate cyclase activator activity / Ras activation upon Ca2+ influx through NMDA receptor / regulation of heart rate / nitric-oxide synthase regulator activity / protein serine/threonine kinase activator activity / sarcomere / FCERI mediated Ca+2 mobilization / FCGR3A-mediated IL10 synthesis / VEGFR2 mediated vascular permeability / positive regulation of peptidyl-threonine phosphorylation / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / VEGFR2 mediated cell proliferation / regulation of cytokinesis / positive regulation of nitric-oxide synthase activity / Translocation of SLC2A4 (GLUT4) to the plasma membrane / spindle microtubule / mitochondrial membrane / RAF activation / positive regulation of receptor signaling pathway via JAK-STAT / Transcriptional activation of mitochondrial biogenesis / positive regulation of protein serine/threonine kinase activity / Stimuli-sensing channels / synaptic vesicle membrane / spindle pole / cellular response to type II interferon / response to calcium ion
Similarity search - Function
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
Similarity search - Domain/homology
2-BROMO-2-CHLORO-1,1,1-TRIFLUOROETHANE / Calmodulin-1 / Calmodulin-3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / simulated annealing, CHARMm22 energy minimization
Model detailsclosest to the average, model 1
AuthorsJuranic, N. / Macura, S. / Simeonov, M.V. / Jones, K.A. / Penheiter, A.R. / Hock, T.J. / Streiff, J.H.
CitationJournal: J. Serb. Chem. Soc. / Year: 2013
Title: Halothane binds to druggable sites in the [Ca2+]4-calmodulin (CaM) complex, but does not inhibit [Ca2+]4-CaM activation of kinase.
Authors: Juranic, N.O. / Jones, K.A. / Penheiter, A.R. / Hock, T.J. / Streiff, J.H.
History
DepositionFeb 17, 2010Deposition site: BMRB / Processing site: RCSB
Revision 1.0Mar 9, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Dec 11, 2013Group: Database references
Revision 1.3Feb 5, 2020Group: Data collection / Database references / Other
Category: database_2 / pdbx_database_status / pdbx_nmr_spectrometer
Item: _pdbx_database_status.status_code_cs / _pdbx_nmr_spectrometer.model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Calmodulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)8,7324
Polymers8,4541
Non-polymers2783
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)5 / 50structures with the lowest energy
RepresentativeModel #1closest to the average

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Components

#1: Protein Calmodulin / / CaM


Mass: 8454.353 Da / Num. of mol.: 1 / Fragment: N-TERMINAL DOMAIN EF-hands 1 and 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: CALM, CALM1, CALM2, CALM3, CALML2, CAM, CAM1, CAM2, CAM3, CAMB, CAMC, CAMIII
Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3)-pLysS / References: UniProt: P62158, UniProt: P0DP23*PLUS
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-HLT / 2-BROMO-2-CHLORO-1,1,1-TRIFLUOROETHANE / Halothane


Mass: 197.382 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2HBrClF3

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
Details: The mechanism(s) of volatile anesthetic effects are poorly understood. We examined whether VA binding to druggable sites in calmodulin would effect [Ca2+]4-CaM dependent activity of enzymes. ...Details: The mechanism(s) of volatile anesthetic effects are poorly understood. We examined whether VA binding to druggable sites in calmodulin would effect [Ca2+]4-CaM dependent activity of enzymes. We used high resolution NMR spectroscopy to determine the structure of the halothane [Ca2+]4-CaM complex, determining that the halothane molecules bind in the druggable sites. We used fluorescence assays to determine that VA mediate [Ca2+]4-CaM activation of smMLCK, but not the kd of [Ca2+]4-CaM binding to skMLCK. These results suggest that VA do not mediate [Ca2+]4-CaM dependent MLCK activity via direct interactions with druggable sites on [Ca2+]4-CaM.
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1113D HNCO
1213D HNCA
1313D HN(CA)CB
1413D HBHA(CO)NH
1513D H(CCO)NH
1613D (H)CCH-TOCSY
1713D 1H-15N NOESY
1813D 1H-13C NOESY
1923D HNHA
11013D (H)CCH-COSY
11123D 1H-15N NOE

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Sample preparation

Details
Solution-IDContentsSolvent system
12 mM [U-99% 13C; U-99% 15N] CALMODULIN, 20 mM CALCIUM ION, 20 mM N-{[2-({[1-(4-CARBOXYBUTANOYL)AMINO]-2-PHENYLETHYL}-HYDROXYPHOSPHINYL)OXY]ACETYL}-2-PHENYLETHYLAMINE, 95% H2O/5% D2O95% H2O/5% D2O
22 mM [U-99% 15N] CALMODULIN, 20 mM CALCIUM ION, 20 mM N-{[2-({[1-(4-CARBOXYBUTANOYL)AMINO]-2-PHENYLETHYL}-HYDROXYPHOSPHINYL)OXY]ACETYL}-2-PHENYLETHYLAMINE, 95% H2O/5% D2O95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
2 mMCALMODULIN-1[U-99% 13C; U-99% 15N]1
20 mMCALCIUM ION-21
20 mMN-{[2-({[1-(4-CARBOXYBUTANOYL)AMINO]-2-PHENYLETHYL}-HYDROXYPHOSPHINYL)OXY]ACETYL}-2-PHENYLETHYLAMINE-31
2 mMCALMODULIN-4[U-99% 15N]2
20 mMCALCIUM ION-52
20 mMN-{[2-({[1-(4-CARBOXYBUTANOYL)AMINO]-2-PHENYLETHYL}-HYDROXYPHOSPHINYL)OXY]ACETYL}-2-PHENYLETHYLAMINE-62
Sample conditionsIonic strength: 0.1 / pH: 7.2 / Pressure: ambient / Temperature: 298 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AvanceBrukerAVANCE7001
Bruker AvanceBrukerAVANCE5002

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Processing

NMR software
NameVersionDeveloperClassification
X-PLORMSI XPLOR 3.843Brungergeometry optimization
X-PLORMSI XPLOR 3.843Brungerrefinement
RefinementMethod: simulated annealing, CHARMm22 energy minimization / Software ordinal: 1 / Details: 100 step steepest descent - final refinement
NMR constraintsNOE constraints total: 735 / NOE intraresidue total count: 314 / NOE long range total count: 128 / NOE medium range total count: 131 / NOE sequential total count: 157 / Hydrogen bond constraints total count: 74 / Protein chi angle constraints total count: 60 / Protein other angle constraints total count: 74 / Protein phi angle constraints total count: 75 / Protein psi angle constraints total count: 74
NMR representativeSelection criteria: closest to the average
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 50 / Conformers submitted total number: 5

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