Correlation of Molecular and Functional Effects of Mutations in Cardiac Troponin T Linked to Familial Hypertrophic Cardiomyopathy AN INTEGRATIVE IN SILICO/IN VITRO APPROACH
Correlation of Molecular and Functional Effects of Mutations in Cardiac Troponin T Linked to Familial Hypertrophic Cardiomyopathy AN INTEGRATIVE IN SILICO/IN VITRO APPROACH
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DOI：
10.1074/jbc.m111.257436
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发表时间：
2012-04-27
影响因子：
4.8
通讯作者：
Tardiff, Jil C.
中科院分区：
文献类型：
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作者：
Manning, Edward P.;Guinto, Pia J.;Tardiff, Jil C.
在导致家族性肥厚型心肌病的所有已知 cTnT 突变中，近 70% 属于对 cTn-Tm 结合至关重要的 TNT1 区域。该结构域的高分辨率结构尚未确定，信息的缺乏阻碍了结构功能分析。在当前的研究中，采用耦合计算实验方法，使用调节体外运动测定（R-IVM）将 cTnT 动态变化与基本功能相关联。使用根据弯曲坐标计算力的计算机方法来精确识别所提出的cTnT“铰链”区域内残基105和106处的弯曲力的减小。在多种功能特性中观察到显着的功能变化，包括钙活化协同性、滑动速度的钙敏感性和最大滑动速度的降低。计算和实验结果的相关性揭示了 TNT1 的灵活性与细丝钙激活的协同性之间的关联，其中灵活性的增加导致协同性的降低。对 TNT1 区域一级序列的进一步分析揭示了由 R92W 和 R92L 突变改变的保守带电 TNT1 残基的独特模式，并且可能代表调节该中心功能域的潜在“结构”。这些数据为进一步整合计算机/体外方法提供了一个框架，该框架可以扩展到高通量预测筛选，以克服当前细丝心肌病分子表型与基因型联系的结构限制。
Nearly 70% of all of the known cTnT mutations that cause familial hypertrophic cardiomyopathy fall within the TNT1 region that is critical to cTn-Tm binding. The high resolution structure of this domain has not been determined, and this lack of information has hindered structure-function analysis. In the current study, a coupled computational experimental approach was employed to correlate changes in cTnT dynamics to basic function using the regulated in vitro motility assay (R-IVM). An in silico approach to calculate forces in terms of a bending coordinate was used to precisely identify decreases in bending forces at residues 105 and 106 within the proposed cTnT "hinge" region. Significant functional changes were observed in multiple functional properties, including a decrease in the cooperativity of calcium activation, the calcium sensitivity of sliding speed, and maximum sliding speed. Correlation of the computational and experimental findings revealed an association between TNT1 flexibility and the cooperativity of thin filament calcium activation where an increase in flexibility led to a decrease in cooperativity. Further analysis of the primary sequence of the TNT1 region revealed a unique pattern of conserved charged TNT1 residues altered by the R92W and R92L mutations and may represent the underlying "structure" modulating this central functional domain. These data provide a framework for further integrated in silico/in vitro approaches that may be extended into a high-throughput predictive screen to overcome the current structural limitations in linking molecular phenotype to genotype in thin filament cardiomyopathies.