Structural studies of interactions between cardiac troponin I and actin in regulated thin filament using Förster resonance energy transfer.
Structural studies of interactions between cardiac troponin I and actin in regulated thin filament using Förster resonance energy transfer.
复制标题
使用 Förster 共振能量转移对调节细丝中心肌肌钙蛋白 I 和肌动蛋白之间的相互作用进行结构研究。
DOI：
10.1021/bi801492x
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Dong,Wen-Ji
中科院分区：
文献类型：
--
作者：
Xing,Jun;Chinnaraj,Mathivanan;Zhang,Zhihong;Cheung,HerbertC;Dong,Wen-Ji
Ca2+诱导的心肌肌钙蛋白I （cTnI）和肌动蛋白之间的相互作用在心肌收缩和舒张的调节中起关键作用。在本报告中，我们研究了这种相互作用的变化，以响应肌凝蛋白S1和肌动蛋白之间的强交叉桥形成和重构细丝中cTnI的PKA磷酸化。通过测量连接cTnI的131、151、160、167、188和210残基的荧光供体5-（碘乙酰氨基乙基）氨基萘磺酸（AEDANS）与连接actin的半胱氨酸374的非荧光受体4-（二甲氨基）苯基偶氮苯基-4′-马来酰亚胺（DABM）之间的Förster共振能量转移（FRET）来监测相互作用。FRET距离测量显示，结合Ca2+诱导从肌动蛋白到cTnI位点的距离大幅增加，表明Ca2+触发cTnI从肌动蛋白分离。在结合Ca2+存在的情况下，强结合的肌球蛋白S1诱导了这些距离的额外增加。这两种配体诱导的增加是相互独立的。这两步距离的变化提供了cTnI和肌动蛋白界面结构变化与细丝调节肌肉收缩和松弛的三态模型的直接联系。当cTnC通过12残基Ca2+结合环内关键残基的突变失活时，强结合的S1单独诱导了距离的增加，尽管纤维丝不再结合调控Ca2+。这些结果表明，单独结合Ca2+或强结合S1可以部分激活细丝，但完全激活需要结合Ca2+和强结合S1。FRET距离的分布揭示了不同生化状态下cTnI不同区域相关的不同结构动力学。第二个肌动蛋白结合区似乎比抑制/调节区更坚硬。在Mg2+状态下，调控区比抑制区更灵活，在Ca2+状态下，抑制区变得更灵活。PKA在不同生化状态下磷酸化了cTnI从肌动蛋白到cTnI残基131的Ser23和Ser24位点的距离2.2−5.2 Å，并缩小了从肌动蛋白到cTnI抑制和调节区域的距离分布。观察到的磷酸化效应可能是由于磷酸化的n端片段和cTnI抑制区域的分子内相互作用。
The Ca2+-induced interaction between cardiac troponin I (cTnI) and actin plays a key role in the regulation of cardiac muscle contraction and relaxation. In this report we have investigated changes of this interaction in response to strong cross-bridge formation between myosin S1 and actin and PKA phosphorylation of cTnI within reconstituted thin filament. The interaction was monitored by measuring Förster resonance energy transfer (FRET) between the fluorescent donor 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid (AEDANS) attached to the residues 131, 151, 160 167, 188, and 210 of cTnI and the nonfluorescent acceptor 4-(dimethylamino)phenylazophenyl-4′-maleimide (DABM) attached to cysteine 374 of actin. The FRET distance measurements showed that bound Ca2+induced large increases in the distances from actin to the cTnI sites, indicating a Ca2+-triggered separation of cTnI from actin. Strongly bound myosin S1 induced additional increases in these distances in the presence of bound Ca2+. The two ligand-induced increases were independent of each other. These two-step changes in distances provide a direct link of structural changes at the interface between cTnI and actin to the three-state model of thin filament regulation of muscle contraction and relaxation. When cTnC was inactivated through mutations of key residues within the 12-residue Ca2+-binding loop, strongly bound S1 alone induced increases in the distances in spite of the fact that the filaments no longer bound regulatory Ca2+. These results suggest bound Ca2+or strongly bound S1 alone can partially activate thin filament, but full activation requires both bound Ca2+and strongly bound S1. The distributions of the FRET distances revealed different structural dynamics associated with different regions of cTnI in different biochemical states. The second actin-binding region appears more rigid than the inhibitory/regulatory region. In the Mg2+state, the regulatory region appears more flexible than the inhibitory region, and in the Ca2+state the inhibitory region becomes more flexible. PKA phosphorylation of cTnI at Ser23 and Ser24 distance from actin to cTnI residue 131 by 2.2−5.2 Å in different biochemical states and narrowed the distributions of the distances from actin to the inhibitory and regulatory regions of cTnI. The observed phosphorylation effects are likely due to an intramolecular interaction of the phosphorylated N-terminal segment and the inhibitory region of cTnI.