Interaction of cardiac troponin C with Ca2+ sensitizer EMD 57033 and cardiac troponin I inhibitory peptide
Interaction of cardiac troponin C with Ca2+ sensitizer EMD 57033 and cardiac troponin I inhibitory peptide
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DOI：
10.1021/bi000473i
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发表时间：
2000-08-01
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：
Li, MX;Spyracopoulos, L;Sykes, BD
Ca 2+与心肌肌钙蛋白C（cTnC）结合触发心肌收缩。在患病的心脏中，心肌通常对Ca 2+不敏感，导致心脏收缩力减弱。心肌对Ca ~（2+）敏感的化合物在治疗心力衰竭方面具有潜在的治疗价值。噻二嗪酮衍生物EMD 57033是一种已鉴定的“钙增敏剂”，cTnC是该药物的潜在靶点。在这项工作中，我们使用2D {H-1，N-15}-HSQC NMR光谱来监测EMD 57033与Ca 2+饱和状态下的cTnC的结合。通过对cTnC结构的化学位移变化进行作图，发现EMD 57033与cTnC的C结构域结合。为了检测EMD 57033是否与心肌肌钙蛋白I（cTnI）竞争cTnC并干扰其抑制功能，我们检测了cTnC与抑制性cTnI肽的相互作用（残基128-147，cIp）。在存在cIp的情况下，也用EMD 57033滴定cTnC。结果表明，尽管药物和cIp都与cTnC的C结构域相互作用，但它们不相互置换，表明两个靶标的非竞争性结合位点。结合位点的详细化学位移图谱显示，包含螺旋G-环IV-螺旋I-I的区域更受EMD 57033的影响，而位于螺旋E-环III-螺旋F上的残基和位点III和IV之间的接头更受cIp的影响。1，在不存在和存在cIp的情况下，药物的结合亲和力分别为8.0 +/-1.8和7.4 +/-4.8 μ M，而在不存在和存在EMD 57033的情况下，肽的那些分别为78.2 ± 10.3和99.2 ± 30.0 μ M。这些结果表明，EMD 57033可能发挥其正性肌力作用不是通过直接增强Ca ~（2+）与cTnC的Ca ~（2+）调节位点的结合，而是通过与cTnC的结构域结合，调节cTnC与其他细丝蛋白之间的相互作用，增加收缩系统的表观Ca ~（2+）敏感性。
The binding of Ca2+ to cardiac troponin C (cTnC) triggers contraction in cardiac muscle. In diseased heart, the myocardium is often desensitized to Ca2+, leading to weak cardiac contractility. Compounds that can sensitize cardiac muscle to Ca2+ would have potential therapeutic value in treating heart failure. The thiadiazinone derivative EMD 57033 is an identified 'Ca2+ sensitizer', and cTnC is a potential target of the drug. In this work, we used 2D {H-1, N-15}-HSQC NMR spectroscopy to monitor the binding of EMD 57033 to cTnC in the Ca2+-saturated state. By mapping the chemical shift changes to the structure of cTnC, EMD 57033 is found to bind to the C-domain of cTnC, To test whether EMD 57033 competes with cardiac TnI (cTnI) for cTnC and interferes with the inhibitory function, we examined the interaction of cTnC with an inhibitory cTnI peptide (residues 128-147, cIp) in the absence and presence of EMD 57033, respectively. cTnC was also titrated with EMD 57033 in the presence of cIp, The results show that although both the drug and cIp interact with the C-domain of cTnC, they do not displace each other, suggesting noncompetitive binding sites for the two targets. Detailed chemical shift mapping of the binding sites reveals that the regions encompassing helix G-loop IV-helix I-I are more affected by EMD 57033, while residues located on helix E-loop III-helix F and the linker between sites III and IV are more affected by cIp, In both cases, the binding stoichiometry is 1:1, The binding affinities for the drug are 8.0 +/- 1.8 and 7.4 +/- 4.8 mu M in the absence and presence of cIp, respectively, while those for the peptide are 78.2 +/- 10.3 and 99.2 +/- 30.0 mu M in the absence and presence of EMD 57033, respectively. These findings suggest that EMD 57033 may exert its positive inotropic effect by not directly enhancing Ca2+ binding to the Ca2+ regulatory site of cTnC, but by binding to the structural domain of cTnC, modulating the interaction between cTnC and other thin filament proteins, and increasing the apparent Ca2+ sensitivity of the contractile system.