Involvement of Conserved, Acidic Residues in the N-Terminal Domain of Troponin C in Calcium-Dependent Regulation

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• 作者：Tomoyoshi Kobayashi ; Xinmei Zhao ; Robert Wade ; and John H. Collins
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：April 27, 1999
• 年：1999
• 卷：38
• 期：17
• 页码：5386 - 5391
• 全文大小：168K
• 年卷期：v.38,no.17(April 27, 1999)
• ISSN：1520-4995

文摘

We have mutated eight conserved, charged amino acid residues in the N-terminal, regulatorydomain of troponin C (TnC) so we could investigate their role in troponin-linked Ca²⁺ regulation ofmuscle contraction. These residues surround a hydrophobic pocket in the N-terminal domain of TnCwhich, when Ca²⁺ binds to regulatory sites in this domain, is exposed and interacts with the inhibitoryregion of troponin I (TnI). We constructed three double mutants (E53A/E54A, E60A/E61A, and E85A/D86A) and two single mutants (R44A and R81A) of rabbit fast skeletal muscle troponin C (TnC) inwhich the charged residues were replaced with neutral alanines. All five of these mutants retained TnC'sability to bind TnI in a Ca²⁺-dependent manner, to neutralize TnI's inhibition of actomyosin S1 ATPaseactivity, and to form a ternary complex with TnI and troponin T (TnT). Ternary complexes formed withTnC(R44A) or TnC(R81A) regulated actomyosin S1 ATPase activity normally, with TnI-based inhibitionin the absence of Ca²⁺ and TnT-based activation in the presence of Ca²⁺. TnC(E53A/E54A) and TnC(E85A/D86A) interacted weakly with TnT, as judged by native gel electrophoresis. Ternary complexesformed with these mutants inhibited actomyosin S1 ATPase activity in both the presence and absence ofCa²⁺, and did not undergo Ca²⁺-dependent structural changes in TnI which can be detected by limitedchymotryptic digestion. TnC(E60A/E61A) interacted normally with TnT. Its ternary complex showedCa²⁺-dependent structural changes in TnI, inhibited actomyosin S1 ATPase in the absence of Ca²⁺, butdid not activate ATPase in the presence of Ca²⁺. This is the first demonstration that selective mutation ofTnC can abolish the activating effect of troponin while its inhibitory function is retained. Our resultssuggest the existence of an elaborate network of protein-protein interactions formed by TnI, TnT, andthe N-terminal domain of TnC, all of which are important in the Ca²⁺-dependent regulation of musclecontraction.