Howarth Jack W, Meller Jarek, Solaro R John, Trewhella Jill, Rosevear Paul R
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, Ohio, 45267, USA.
J Mol Biol. 2007 Oct 26;373(3):706-22. doi: 10.1016/j.jmb.2007.08.035. Epub 2007 Aug 22.
We present here the solution structure for the bisphosphorylated form of the cardiac N-extension of troponin I (cTnI(1-32)), a region for which there are no previous high-resolution data. Using this structure, the X-ray crystal structure of the cardiac troponin core, and uniform density models of the troponin components derived from neutron contrast variation data, we built atomic models for troponin that show the conformational transition in cardiac troponin induced by bisphosphorylation. In the absence of phosphorylation, our NMR data and sequence analyses indicate a less structured cardiac N-extension with a propensity for a helical region surrounding the phosphorylation motif, followed by a helical C-terminal region (residues 25-30). In this conformation, TnI(1-32) interacts with the N-lobe of cardiac troponin C (cTnC) and thus is positioned to modulate myofilament Ca2+-sensitivity. Bisphosphorylation at Ser23/24 extends the C-terminal helix (residues 21-30) which results in weakening interactions with the N-lobe of cTnC and a re-positioning of the acidic amino terminus of cTnI(1-32) for favorable interactions with basic regions, likely the inhibitory region of cTnI. An extended poly(L-proline)II helix between residues 11 and 19 serves as the rigid linker that aids in re-positioning the amino terminus of cTnI(1-32) upon bisphosphorylation at Ser23/24. We propose that it is these electrostatic interactions between the acidic amino terminus of cTnI(1-32) and the basic inhibitory region of troponin I that induces a bending of cTnI at the end that interacts with cTnC. This model provides a molecular mechanism for the observed changes in cross-bridge kinetics upon TnI phosphorylation.
我们在此展示肌钙蛋白I心脏N端延伸区双磷酸化形式（cTnI(1 - 32)）的溶液结构，此前该区域尚无高分辨率数据。利用此结构、心脏肌钙蛋白核心的X射线晶体结构以及源自中子对比变化数据的肌钙蛋白各组分的统一密度模型，我们构建了肌钙蛋白的原子模型，该模型展示了双磷酸化诱导的心脏肌钙蛋白的构象转变。在无磷酸化的情况下，我们的核磁共振数据和序列分析表明，心脏N端延伸区结构较松散，围绕磷酸化基序有形成螺旋区域的倾向，随后是螺旋状的C端区域（第25 - 30位氨基酸残基）。在此构象中，TnI(1 - 32)与心脏肌钙蛋白C（cTnC）的N叶相互作用，因此处于调节肌丝Ca2 +敏感性的位置。Ser23/24位点的双磷酸化使C端螺旋（第21 - 30位氨基酸残基）延伸，这导致与cTnC的N叶相互作用减弱，以及cTnI(1 - 32)酸性氨基末端重新定位以与碱性区域（可能是cTnI的抑制区域）进行有利的相互作用。第11至19位氨基酸残基之间延伸的聚（L - 脯氨酸）II螺旋作为刚性连接体，有助于在Ser23/24位点双磷酸化时重新定位cTnI(1 - 32)的氨基末端。我们提出，正是cTnI(1 - 32)的酸性氨基末端与肌钙蛋白I的碱性抑制区域之间的这些静电相互作用，诱导了cTnI在与cTnC相互作用的末端发生弯曲。该模型为观察到的TnI磷酸化后横桥动力学变化提供了分子机制。
