The bindin of W7, an inhibitor of striated muscle contraction, to cardiac troponin C
The bindin of W7, an inhibitor of striated muscle contraction, to cardiac troponin C
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DOI：
10.1021/bi051583y
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发表时间：
2005-12-06
期刊：
影响因子：
2.9
通讯作者：
Sykes, BD
中科院分区：
文献类型：
--
作者：
Hoffman, RMB;Li, MX;Sykes, BD
W7是一种特性良好的钙调素拮抗剂。降低心肌纤维的最大张力和ATP水解速率。心肌肌钙蛋白C（cTnC）以前被认为是肌丝中W7的重要机制靶点。二维NMR光谱（{H-1，N-15}-和{H-1，C-13}-HSQC）用于监测W7与cTnC的Ca 2+依赖性结合。cTnC滴定（.）3Ca（2+）与W7的结合表明与蛋白的两个结构域结合。我们检查了W7与cTnC的分离结构域的结合以简化光谱分析。在C-末端结构域（cCTnC（.）2Ca（2+）），来自一个残基子集的光谱峰变化不均匀，不能很好地描述为单位点结合。cCTnC（.）的全局拟合2Ca（2+）滴定数据与双位点顺序结合模型（同时拟合47个残基）的拟合结果显示，单一结合状态的解离常数（Kd 1）为0.85-0.91 mM，第二个解离常数拟合为3.40-3.65 mM（>= 4 x Kd 1）。N-末端结构域（cNTnC（.）Ca（2+））整体拟合至具有0.15-0.30 mM的Kd（41个残基拟合）的单位点结合模型。这些数据与W7与每个结构域的主要疏水口袋结合一致，协调负责连接cTnI的侧链。当在肌纤维中时，W7可能与cTnI竞争cTnC上的靶位点。
W7 is a well-characterized calmodulin antagonist. It decreases the maximal tension and rate of ATP hydrolysis in cardiac Muscle fibers. Cardiac troponin C (cTnC) has been previously implicated as the mechanistically significant target for W7 in the myofilarnent. Two-dimensional NMR spectra ({H-1, N-15}- and {H-1,C-13}-HSQCs) were used to monitor the Ca2+-dependent binding of W7 to cTnC. Titration of cTnC(.)3Ca(2+) with W7 indicated binding to both domains of the protein. We examined the binding of W7 to the separated domains of cTnC to simplify the spectral analysis. In the titration of the C-terminal domain (cCTnC(.)2Ca(2+)), the spectral peaks originating from a subset of residues changed nonuniformly, and Could not be well-described as single-site binding. A global fit of the cCTnC(.)2Ca(2+) titration data to a two-site, sequential binding model (47 residues simultaneously fit) yielded a dissociation constant (Kd1) of 0.85-0.91 mM for the singly bound state, with the second dissociation constant fit to 3.40-3.65 mM (>= 4 x Kd1). The titration data for the N-terminal domain (cNTnC(.)Ca(2+)) was globally fit to single-site binding model with a Kd of 0.15-0.30 mM (41 residues fit). The data are consistent with W7 binding to each domain's major hydrophobic pocket, coordinating side chains responsible for liganding cTnI. When in muscle fibers, W7 may compete with cTnI for target sites on cTnC.