Potluri Phani R, Chamoun Jean, Cooke James A, Badr Myriam, Guse Joanna A, Rayes Roni, Cordina Nicole M, McCamey Dane, Fajer Piotr G, Brown Louise J
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, New South Wales 2109, Australia.
Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA.
J Struct Biol. 2017 Dec;200(3):376-387. doi: 10.1016/j.jsb.2017.08.007. Epub 2017 Aug 31.
The absence of a crystal structure of the calcium free state of the cardiac isoform of the troponin complex has hindered our understanding of how the simple binding of Ca triggers conformational changes in troponin which are then propagated to enable muscle contraction. Here we have used continuous wave (CW) and Double Electron-Electron Resonance (DEER) pulsed EPR spectroscopy to measure distances between TnI and TnC to track the movement of the functionally important regulatory 'switch' region of cardiac Tn. Spin labels were placed on the switch region of Troponin I and distances measured to Troponin C. Under conditions of high Ca, the interspin distances for one set (TnI151/TnC84) were 'short' (9-10Å) with narrow distance distribution widths (3-8Å) indicating the close interaction of the switch region with the N-lobe of TnC. Additional spin populations representative of longer interspin distances were detected by DEER. These longer distance populations, which were ∼16-19Å longer than the short distance populations, possessed notably broader distance distribution widths (14-29Å). Upon Ca removal, the interspin population shifted toward the longer distances, indicating the release of the switch region from TnC and an overall increase in disorder for this region. Together, our results suggest that under conditions of low Ca, the close proximity of the TnI switch region to TnC in the cardiac isoform is necessary for promoting the interaction between the regulatory switch helix with the N-lobe of cardiac Troponin C, which, unlike the skeletal isoform, is largely in a closed conformation.
肌钙蛋白复合物心脏亚型的无钙状态缺乏晶体结构，这阻碍了我们对钙离子的简单结合如何触发肌钙蛋白构象变化的理解，而这种构象变化随后会传播以实现肌肉收缩。在这里，我们使用连续波（CW）和双电子-电子共振（DEER）脉冲EPR光谱来测量肌钙蛋白I（TnI）和肌钙蛋白C（TnC）之间的距离，以追踪心脏Tn功能上重要的调节“开关”区域的运动。自旋标记物被放置在肌钙蛋白I的开关区域，并测量到肌钙蛋白C的距离。在高钙条件下，一组（TnI151/TnC84）的自旋间距离为“短”（9-10Å），距离分布宽度较窄（3-8Å），表明开关区域与TnC的N叶紧密相互作用。通过DEER检测到了代表较长自旋间距离的额外自旋群体。这些较长距离的群体比短距离群体长约16-19Å，其距离分布宽度明显更宽（14-29Å）。去除钙离子后，自旋群体向更长距离移动，表明开关区域从TnC释放，并且该区域的无序度总体增加。总之，我们的结果表明，在低钙条件下，心脏亚型中TnI开关区域与TnC的紧密接近对于促进调节开关螺旋与心脏肌钙蛋白C的N叶之间的相互作用是必要的，与骨骼肌亚型不同，心脏肌钙蛋白C在很大程度上处于封闭构象。
