title: A cross-linking study of the N-terminal extension of human cardiac troponin I.
abstract: Phosphorylation of the unique N-terminal extension of cardiac troponin I (TnI) by PKA modulates Ca(2+) release from the troponin complex. The mechanism by which phosphorylation affects Ca(2+) binding, however, remains unresolved. To investigate this question, we have studied the interaction of a fragment of TnI consisting of residues 1-64 (I1-64) with troponin C (TnC) by isothermal titration microcalorimetry and cross-linking. I1-64 binds extremely tightly to the C-terminal domain of TnC and weakly to the N-terminal domain. Binding to the N-domain is weakened further by phosphorylation. Using the heterobifunctional cross-linker benzophenone-4-maleimide and four separate cysteine mutants of I1-64 (S5C, E10C, I18C, R26C), we have probed the protein-protein interactions of the N-terminal extension. All four I1-64 mutants cross-link to the N-terminal domain of TnC. The cross-linking is enhanced by Ca(2+) and reduced by phosphorylation. By introducing the same monocysteine mutations into full-length TnI, we were able to probe the environment of the N-terminal extension in intact troponin. We find that the full length of the extension lies in close proximity to both TnC and troponin T (TnT). Ca(2+) enhances the cross-linking to TnC. Cross-linking to both TnC and TnT is reduced by prior phosphorylation of the TnI. In binary complexes the mutant TnIs cross-link to both the isolated TnC N-domain and whole TnC. Cyanogen bromide digestion of the covalent TnI-TnC complex formed from intact troponin demonstrates that cross-linking is predominantly to the N-terminal domain of TnC.
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, UK.
authors.forename: Douglas G
authors.initials: DG
authors.lastname: Ward
authors.forename: Susan M
authors.initials: SM
authors.lastname: Brewer
authors.forename: Michael P
authors.initials: MP
authors.lastname: Cornes
authors.forename: Ian P
authors.initials: IP
authors.lastname: Trayer
mesh_terms: D019943:Amino Acid Substitution; D001577:Benzophenones / Q000737:chemistry; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology; D002151:Calorimetry / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003488:Cyanogen Bromide / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008301:Maleimides / Q000737:chemistry; D009206:Myocardium / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D013997:Time Factors; D019209:Troponin C / Q000737:chemistry / Q000378:metabolism; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001577:Benzophenones; D003432:Cross-Linking Reagents; D008301:Maleimides; D011994:Recombinant Proteins; D019209:Troponin C; D019210:Troponin I; C046109:benzophenone-4-maleimide; D003545:Cysteine; D003488:Cyanogen Bromide; D002118:Calcium