title: Characterization of the interaction between the N-terminal extension of human cardiac troponin I and troponin C.
abstract: The N-terminal extension of cardiac troponin I (TnI) is bisphosphorylated by protein kinase A in response to beta-adrenergic stimulation. How this signal is transmitted between TnI and troponin C (TnC), resulting in accelerated Ca(2+) release, remains unclear. We recently proposed that the unphosphorylated extension interacts with the N-terminal domain of TnC stabilizing Ca(2+) binding and that phosphorylation prevents this interaction. We now use (1)H NMR to study the interactions between several N-terminal fragments of TnI, residues 1-18 (I1-18), residues 1-29 (I1-29), and residues 1-64 (I1-64), and TnC. The shorter fragments provide unambiguous information on the N-terminal regions of TnI that interact with TnC: I1-18 does not bind to TnC whereas the C-terminal region of unphosphorylated I1-29 does bind. Bisphosphorylation greatly weakens this interaction. I1-64 contains the phosphorylatable N-terminal extension and a region that anchors I1-64 to the C-terminal domain of TnC. I1-64 binding to TnC influences NMR signals arising from both domains of TnC, providing evidence that the N-terminal extension of TnI interacts with the N-terminal domain of TnC. TnC binding to I1-64 broadens NMR signals from the side chains of residues immediately C-terminal to the phosphorylation sites. Binding of TnC to bisphosphorylated I1-64 does not broaden these NMR signals to the same extent. Circular dichroism spectra of I1-64 indicate that bisphosphorylation does not produce major secondary structure changes in I1-64. We conclude that bisphosphorylation of cardiac TnI elicits its effects by weakening the interaction between the region of TnI immediately C-terminal to the phosphorylation sites and TnC either directly, due to electrostatic repulsion, or via localized conformational changes.
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Ward
authors.forename: Susan M
authors.initials: SM
authors.lastname: Brewer
authors.forename: Melanie J
authors.initials: MJ
authors.lastname: Calvert
authors.forename: Clare E
authors.initials: CE
authors.lastname: Gallon
authors.forename: Yuan
authors.initials: Y
authors.lastname: Gao
authors.forename: Ian P
authors.initials: IP
authors.lastname: Trayer
mesh_terms: D000409:Alanine / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D055672:Static Electricity; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019210:Troponin I / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D019209:Troponin C; D019210:Troponin I; D018698:Glutamic Acid; D000409:Alanine; D002118:Calcium