Baryshnikova Olga K, Robertson Ian M, Mercier Pascal, Sykes Brian D
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Biochemistry. 2008 Oct 14;47(41):10950-60. doi: 10.1021/bi801165c. Epub 2008 Sep 20.
NMR spectroscopy has been employed to elucidate the molecular consequences of the DCM G159D mutation on the structure and dynamics of troponin C, and its interaction with troponin I (TnI). Since the molecular effects of human mutations are often subtle, all NMR experiments were conducted as direct side-by-side comparisons of the wild-type C-domain of troponin C (cCTnC) and the mutant protein, G159D. With the mutation, the affinity toward the anchoring region of cTnI (cTnI 34-71) was reduced ( K D = 3.0 +/- 0.6 microM) compared to that of the wild type ( K D < 1 microM). Overall, the structure and dynamics of the G159D.cTnI 34-71 complex were very similar to those of the cCTnC.cTnI 34-71 complex. There were, however, significant changes in the (1)H, (13)C, and (15)N NMR chemical shifts, especially for the residues in direct contact with cTnI 34-71, and the changes in NOE connectivity patterns between the G159D.cTnI 34-71 and cCTnC.cTnI 34-71 complexes. Thus, the most parsimonious hypothesis is that the development of disease results from the poor anchoring of cTnI to cCTnC, with the resulting increase in the level of acto-myosin inhibition in agreement with physiological data. Another possibility is that long-range electrostatic interactions affect the binding of the inhibitory and switch regions of cTnI (cTnI 128-147 and cTnI 147-163) and/or the cardiac specific N-terminus of cTnI (cTnI 1-29) to the N-domain of cTnC. These important interactions are all spatially close in the X-ray structure of the cardiac TnC core.
核磁共振光谱已被用于阐明DCM G159D突变对肌钙蛋白C的结构和动力学及其与肌钙蛋白I（TnI）相互作用的分子影响。由于人类突变的分子效应往往很微妙，所有核磁共振实验都是作为肌钙蛋白C的野生型C结构域（cCTnC）和突变蛋白G159D的直接并排比较进行的。发生突变后，与野生型相比，对cTnI锚定区域（cTnI 34 - 71）的亲和力降低（KD = 3.0 +/- 0.6 microM）（野生型KD < 1 microM）。总体而言，G159D.cTnI 34 - 71复合物的结构和动力学与cCTnC.cTnI 34 - 71复合物非常相似。然而，（1）H、（13）C和（15）N核磁共振化学位移有显著变化，特别是与cTnI 34 - 71直接接触的残基，以及G159D.cTnI 34 - 71和cCTnC.cTnI 34 - 71复合物之间的核Overhauser效应（NOE）连接模式的变化。因此，最合理的假设是疾病的发生是由于cTnI与cCTnC的锚定不佳，导致肌动球蛋白抑制水平升高，这与生理学数据一致。另一种可能性是远程静电相互作用影响cTnI的抑制区和开关区（cTnI 128 - 147和cTnI 147 - 163）和/或cTnI的心脏特异性N端（cTnI 1 - 29）与cTnC的N结构域的结合。在心脏肌钙蛋白C核心的X射线结构中，这些重要相互作用在空间上都很接近。
