Ward Douglas G, Brewer Susan M, Cornes Michael P, Trayer Ian P
School of Biosciences, University of Birmingham, Edgbaston, UK.
Biochemistry. 2003 Sep 2;42(34):10324-32. doi: 10.1021/bi034495r.
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.
蛋白激酶A（PKA）对心肌肌钙蛋白I（TnI）独特的N端延伸区域进行磷酸化修饰，可调节肌钙蛋白复合物中Ca(2+)的释放。然而，磷酸化影响Ca(2+)结合的机制仍未明确。为研究该问题，我们通过等温滴定量热法和交联实验，研究了由1 - 64位残基组成的TnI片段（I1 - 64）与肌钙蛋白C（TnC）的相互作用。I1 - 64与TnC的C端结构域紧密结合，与N端结构域的结合较弱。磷酸化会进一步削弱其与N结构域的结合。使用异双功能交联剂二苯甲酮-4-马来酰亚胺以及I1 - 64的四个不同半胱氨酸突变体（S5C、E10C、I18C、R26C），我们探究了N端延伸区域的蛋白质-蛋白质相互作用。所有四个I1 - 64突变体均与TnC的N端结构域发生交联。Ca(2+)可增强交联作用，而磷酸化则会使其减弱。通过将相同的单半胱氨酸突变引入全长TnI，我们能够探究完整肌钙蛋白中N端延伸区域的环境。我们发现，延伸区域的全长与TnC和肌钙蛋白T（TnT）都非常接近。Ca(2+)增强了与TnC的交联作用。TnI预先磷酸化会减少与TnC和TnT的交联。在二元复合物中，突变的TnI与分离的TnC N结构域和完整的TnC均发生交联。对由完整肌钙蛋白形成的共价TnI-TnC复合物进行溴化氰消化，结果表明交联主要发生在TnC的N端结构域。
