Effects of Calcium Binding and the Hypertrophic Cardiomyopathy A8V Mutation on the Dynamic Equilibrium between Closed and Open Conformations of the Regulatory N-Domain of Isolated Cardiac Troponin C
Effects of Calcium Binding and the Hypertrophic Cardiomyopathy A8V Mutation on the Dynamic Equilibrium between Closed and Open Conformations of the Regulatory N-Domain of Isolated Cardiac Troponin C
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DOI：
10.1021/bi4000172
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发表时间：
2013-03-19
期刊：
影响因子：
2.9
通讯作者：
Brown, Louise J.
中科院分区：
文献类型：
--
作者：
Cordina, Nicole M.;Liew, Chu K.;Brown, Louise J.
肌钙蛋白C(TNC)是肌钙蛋白复合体的钙结合亚单位，负责启动横纹肌收缩以响应钙内流。在骨骼TNC异构体中，钙结合导致TNC调控N-结构域的结构变化，包括从闭合结构状态到开放结构状态的转变，并伴随着肌钙蛋白I(TnI)随后结合的大片疏水斑块的暴露。然而，只有在结合的TnI存在的情况下，才能观察到钙如何启动心脏异构体(CTNC)的N-结构域与TnI亚单位相互作用，因为cTNC调节域的开放构象。在这里，我们使用顺磁驰豫增强(PRE)来表征孤立的cTNC在溶液中从闭合到开放的转变，这是传统的核磁共振方法无法观察到的过程。我们来自分离的cTNC的四个自旋标记的单半胱氨酸结构的前数据表明，钙结合触发N-结构域螺旋向开放状态移动。将Pre数据与封闭到开放的转变模型进行拟合，发现在没有钙的情况下，存在少量具有开放构象的cTNC分子，其水平在钙离子结合时显著增加。这些数据支持一个模型，在该模型中，钙结合在闭合和开放结构状态之间创建动态平衡，以启动cTNC与其靶肽的相互作用。我们还使用Pre数据来评估位于cTNC(A8V)N区域内的一个家族性肥厚型心肌病点突变的结构效应。Pre数据表明，A8V突变扰乱了钙离子开关机制，导致在apo和holo状态下N-结构域构象更加开放。
Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca2+ binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca2+ switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states.