Regulatory Domain Conformational Exchange and Linker Region Flexibility in Cardiac Troponin C Bound to Cardiac Troponin I

Abbott, M.Bret; Gaponenko, Vadim; Abusamhadneh, Ekram; Finley, Natosha L.; Li, Ge; Dvoretsky, Alex; Rance, Mark; Solaro, R. John; Rosevear, Paul R.

doi:10.1074/jbc.m909252199

Cited by 50 publications

(74 citation statements)

References 44 publications

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“…Previously we showed that amide chemical shifts could be utilized to monitor conformation equilibria between open and closed N-lobe substates (16). We see no evidence that the introduction of a negative charge at Ser 43 /Ser 45 of cTnI significantly alters conformational equilibria in the N-lobe of cTnC (Fig.…”

Section: Methodsmentioning

confidence: 82%

“…Binary complexes were judged 1:1 by native gel electrophoresis in the presence of 10 mM CaCl 2 and by sensitivity-enhanced 1 H-15 N HSQC spectra (16). Samples for NMR were ϳ1 mM in Ca 2ϩ -saturated protein in 20 mM Tris d11 , pH 6.8, 100 mM KCl, 5 mM dithiothreitol, 10 mM CaCl 2 , and 10% 2 H 2 O.…”

Section: Methodsmentioning

confidence: 99%

“…Complex Formation and Resonance Assignment-Samples of Ca 2ϩ -saturated [ 2 H, 15 N]cTnC or [ 13 C, 15 N]CcTnC were combined in equimolar amounts with either cTnI(S43D/S45D) or NcTnI(S43D/S45D) to form stable binary complexes as described previously (16). Binary complexes were judged 1:1 by native gel electrophoresis in the presence of 10 mM CaCl 2 and by sensitivity-enhanced 1 H-15 N HSQC spectra (16).…”

Section: Methodsmentioning

confidence: 99%

“…Recombinant Protein Expression and Purification-Isotopically enriched and unlabeled recombinant proteins were expressed, purified, and quantified as described (16,17).…”

Section: Methodsmentioning

confidence: 99%

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Introduction of Negative Charge Mimicking Protein Kinase C Phosphorylation of Cardiac Troponin I

Finley¹,

Rosevear

2004

Journal of Biological Chemistry

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Protein kinase C phosphorylation of cardiac troponin, the Ca 2؉ -sensing switch in muscle contraction, is capable of modulating the response of cardiac muscle to a Ca 2؉ ion concentration. The N-domain of cardiac troponin I contains two protein kinase C phosphorylation sites. Although the physiological consequences of phosphorylation at Ser 43 /Ser 45 are known, the molecular mechanisms responsible for these functional changes have yet to be established. In this work, NMR was used to identify conformational and dynamic changes in cardiac troponin C upon binding a phosphomimetic troponin I, having Ser 43 /Ser 45 mutated to Asp. Chemical shift perturbation mapping indicated that residues in helix G were most affected. Smaller chemical shift changes were observed in residues located in the Ca 2؉ /Mg 2؉ -binding loops. Amide hydrogen/deuterium exchange rates in the C-lobe of troponin C were compared in complexes containing either the wild-type or phosphomimetic N-domain of troponin I. In the presence of a phosphomimetic domain, exchange rates in helix G increased, whereas a decrease in exchange rates for residues mapping to Ca 2؉ /Mg 2؉ -binding loops III and IV was observed. Increased exchange rates are consistent with destabilization of the Thr 129 -Asp 132 helix capping box previously characterized in helix G. The perturbation of helix G and metal binding loops III and IV suggests that phosphorylation alters metal ion affinity and inter-subunit interactions. Our studies support a novel mechanism for protein kinase C signal transduction, emphasizing the importance of C-lobe Ca 2؉ /Mg 2؉ -dependent troponin interactions.Troponin and tropomyosin form the Ca 2ϩ -sensitive switch that regulates striated muscle contraction. Troponin is a ternary assembly of proteins composed of the Ca 2ϩ -binding subunit troponin C (TnC), 1 the inhibitory subunit troponin I (TnI), and the tropomyosin-binding protein troponin T that anchors troponin to the thin filament. Troponin C, a member of the EF-hand family of Ca 2ϩ -binding proteins, contains two globular domains connected by a linker. Each domain of cTnC contains two EF-hand or Ca 2ϩ -binding motifs. The N-lobe contains two lower affinity Ca 2ϩ -binding motifs, sites I and II, that control muscle contraction. Site I is naturally inactive in the cardiac isoform because of several amino acid substitutions and an amino acid insertion (1). Thus, Ca 2ϩ binding at site II in cTnC regulates muscle contraction. The C-lobe contains two high affinity Ca 2ϩ -binding sites, III and IV, which also bind Mg 2ϩ with lower affinity. Interactions between the C-lobe of cTnC and the N-domain of cTnI form the Ca 2ϩ /Mg 2ϩ -dependent cTnC/cTnI interaction site. In addition, the C-lobe of cTnC also interacts tightly with the C terminus of cardiac troponin T (2). These interactions form the core of the troponin complex, tethering all three subunits throughout the contraction cycle.A variety of effectors can modulate the frequency and intensity of myocardial contraction by charge modification upon the p...

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Section: Methodsmentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Recombinant Protein Expression and Purification-Isotopically enriched and unlabeled recombinant proteins were expressed, purified, and quantified as described (16,17).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Introduction of Negative Charge Mimicking Protein Kinase C Phosphorylation of Cardiac Troponin I

Finley¹,

Rosevear

2004

Journal of Biological Chemistry

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“…This indicates that the pathways involved in initiating skeletal and cardiac muscle contraction are structurally very similar; however, the kinetics and thermodynamics of the pathways must differ for the two systems to account for the different physiological behavior of the two muscle types [15]. NMR studies of TnC with various TnI peptides have yielded detailed structural information on the structure of TnC when bound to TnI [16][17][18][19], on the structure of TnI inhibitory peptide [20,21], and on the overall topology of TnC-TnI arrangement [22][23][24][25][26][27][28][29][30][31][32]. The high-resolution structures of TnC-TnI available are the X-ray structure of sTnC·2Ca 2+ ·sTnI [33], the NMR structures of cNTnC·Ca 2+ ·cTnI 147-163 [13], sNTnC(rhodamine)·2Ca 2+ ·sTnI 115-131 [14], and cCTnC·2Ca 2+ ·cTnI 128-147 [34], the X-ray structure of the core domain cardiac troponin complex, cTnC·3Ca 2+ ·cTnI ·cTnT2 182-288 [35], and the X-ray structures of skeletal troponin complex in both the apo and Ca 2+ -state, sTnC·apo·sTnI ·sTnT 156-262 and sTnC·4Ca 2+ ·sTnI ·sTnT [36].…”

Section: Introductionmentioning

confidence: 99%

Interaction of cardiac troponin with cardiotonic drugs: A structural perspective

Robertson

Sykes

2008

Biochemical and Biophysical Research Communications

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Over the forty years since its discovery, many studies have focused on understanding the role of troponin as a myofilament based molecular switch in regulating the Ca 2+ -dependent activation of striated muscle contraction. Recently, studies have explored the role of cardiac troponin as a target for cardiotonic agents. These drugs are clinically useful for treating heart failure, a condition in which the heart is no longer able to pump enough blood to other organs. These agents act via a mechanism that modulates the Ca 2+ -sensitivity of troponin; such a mode of action is therapeutically desirable because intracellular Ca 2+ concentration is not perturbed, preserving the regulation of other Ca 2+ -based signaling pathways. This review describes molecular details of the interaction of cardiac troponin with a variety of cardiotonic drugs. We present recent structural work that has identified the docking sites of several cardiotonic drugs in the troponin C -troponin I interface and discuss their relevance in the design of troponin based drugs for the treatment of heart disease.

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Modulation of Cardiac Myofilament Activity by Protein Phosphorylation

Solaro

2002

Comprehensive Physiology

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Regulatory Domain Conformational Exchange and Linker Region Flexibility in Cardiac Troponin C Bound to Cardiac Troponin I

Cited by 50 publications

References 44 publications

Introduction of Negative Charge Mimicking Protein Kinase C Phosphorylation of Cardiac Troponin I

Introduction of Negative Charge Mimicking Protein Kinase C Phosphorylation of Cardiac Troponin I

Interaction of cardiac troponin with cardiotonic drugs: A structural perspective

Modulation of Cardiac Myofilament Activity by Protein Phosphorylation

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