Dong Wen-Ji, Xing Jun, Ouyang Yexin, An Jianli, Cheung Herbert C
School of Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington 99164; Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, Washington 99164.
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Alabama 35294.
J Biol Chem. 2008 Feb 8;283(6):3424-3432. doi: 10.1074/jbc.M703822200. Epub 2007 Dec 5.
The key events in regulating cardiac muscle contraction involve Ca(2+) binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca(2+) movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca(2+) binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca(2+)-induced opening and closing of the N-domain by Förster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca(2+). L29Q had no effect on the closing rate of the N-domain triggered by release of Ca(2+), but reduced the Ca(2+)-induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca(2+) dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC.
调节心肌收缩的关键事件包括钙离子与肌钙蛋白C（cTnC）结合及从cTnC释放，以及由钙离子移动引发的cTnC和其他细肌丝蛋白的结构变化。据报道，人类cTnC中的单突变L29Q和G159D分别与家族性肥厚型心肌病和扩张型心肌病相关。我们研究了这些单个突变对由钙离子结合和解离引发的cTnC调节性N结构域结构转变的影响。本研究使用了cTnC的双突变体或三突变体，将其与无色氨酸的cTnI和cTnT重组为肌钙蛋白。用1,5 - IAEDANS标记半胱氨酸-51的双突变体cTnC(L12W/N51C)作为对照，通过Förster共振能量转移（FRET）监测钙离子诱导的N结构域的打开和关闭。三突变体包含用1,5 - IAEDANS标记的L12W和N51C，以及L29Q或G159D中的一个。在有无结合钙离子的情况下，这两种突变对色氨酸-12和半胱氨酸-51 - AEDANS之间的平衡距离影响最小。L29Q对钙离子释放引发的N结构域关闭速率没有影响，但降低了钙离子诱导的打开速率。G159D降低了关闭和打开速率。先前的结果表明，cTnI的蛋白激酶A（PKA）磷酸化可显著提高由钙离子解离引发的cTnC N结构域的关闭速率。L29Q或G159D消除了这种速率增强。这些突变改变了cTnC调节性N结构域中参与激活（L29Q）或失活（G159D）的结构转变动力学。这两种突变似乎都对cTnI和cTnC之间的磷酸化信号传导具有拮抗作用。
