Characterization of zero-length cross-links between rabbit skeletal muscle troponin C and troponin I: evidence for direct interaction between the inhibitory region of troponin I and the NH2-terminal, regulatory domain of troponin C.
Characterization of zero-length cross-links between rabbit skeletal muscle troponin C and troponin I: evidence for direct interaction between the inhibitory region of troponin I and the NH2-terminal, regulatory domain of troponin C.
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兔骨骼肌肌钙蛋白 C 和肌钙蛋白 I 之间零长度交联的表征：肌钙蛋白 I 的抑制区和肌钙蛋白 C 的 NH2 末端调节域之间直接相互作用的证据。
DOI：
10.1021/bi00453a041
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Collins,JH
中科院分区：
文献类型：
--
作者：
Leszyk,J;Grabarek,Z;Gergely,J;Collins,JH
马里兰大学医学院生物化学系和医学生物技术中心，马里兰州巴尔的摩 21201，波士顿生物医学研究所肌肉研究部和马萨诸塞州总医院神经病学服务部，波士顿，马萨诸塞州 02114，以及哈佛医学院生物化学和分子药理学系，波士顿，马萨诸塞州 02115 1989 年 6 月 21 日收到；修订稿 1989 年 8 月 21 日收稿 摘要；肌钙蛋白 C (TnC) 和肌钙蛋白 I (Tnl) 之间的相互作用在脊椎动物横纹肌收缩的 Ca2+ 依赖性调节中发挥重要作用。先前试图阐明TnC-Tnl相互作用的分子细节，主要涉及化学修饰的蛋白质或其片段，已经导致了广泛接受的想法，即Tnl的“抑制区”（残基96-116）与TnC的α螺旋片段结合，该α螺旋片段包含非调节性COOH末端结构域中的残基89-100。为了确定这些蛋白质之间其他可能的生理上重要的相互作用，使用 1-乙基-3-[3-(二甲氨基)丙基]碳二亚胺 (EDC) 在兔骨骼肌 TnC 和 TnI 复合物中产生零长度交联。 TnC 用 EDC 和 N-羟基琥珀酰亚胺 (NHS) 活化，然后与等摩尔量的 TnI 混合 [Grabarek, Z., & Gergely, J.(1988) Biophys. J. 53, 392a]。所得交联 TnCXI 用溴化氰、胰蛋白酶和金黄色葡萄球菌 V8 蛋白酶 (SAP) 裂解。通过反相 HPLC 纯化交联肽并通过序列分析进行表征。结果表明，来自TnC的NH 2 末端结构域中的调节Ca 2+ 结合位点II的残基（残基46-78）与跨越残基92-167的Tnl片段形成交联。 Tnl 中交联度最高的残基是位于抑制区的 Lys-105 和 Lys-107。这些结果为 TnC 的 N 末端结构域和 Tnl 的抑制区域之间的相互作用提供了第一个证据。 e Ca2+ 结合 (TnC) 1 和肌钙蛋白抑制 (Tnl) 成分之间相互作用的 Ca2+ 依赖性变化是骨骼肌收缩激活过程中的关键事件之一 [有关评论，请参阅 Leavis 和 Gergely (1984) 以及 Zot 和 Potter (1987)]。为了了解肌肉激活的分子基础，人们付出了很多努力来表征 TnC-Tnl 界面。兔快速骨骼肌 TnC（Collins 等，1973，1977）和 Tnl（Wilkinson & Grand，1975）的氨基酸序列是已知的，并且这些蛋白质已作为在多个实验室进行的广泛结构功能研究的模型。虽然关于 Tnl 的三维结构还有很多需要了解，但对 TnC 序列的分析（Collins 等，1973）预测了四个 Ca2+ 结合位点的位置，指定为 I-IV，从蛋白质的氨基末端到羧基末端。这一预测后来被鸡 (Sundaralingam et al., 1985) 和火鸡 (Herzberg & James, 1985) TnCs 的晶体结构所证实，
Department of Biological Chemistry, School of Medicine, and Medical BiotechnologyCenter of the Maryland Biotechnology Institute, University of Maryland, Baltimore, Maryland 21201, Department of Muscle Research, Boston Biomedical Research Institute, and Neurology Service, Massachusetts General Hospital, Boston, Massachusetts 02114, and Departments of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 Received June 21, 1989; Revised Manuscript Received August 21, 1989 abstract; Interactions between troponin C (TnC) and troponin I (Tnl) play an important role inthe Ca2+-dependent regulation of vertebrate striated muscle contraction. Previous attempts to elucidate the molecular details of TnC-Tnl interactions, mainly involving chemically modified proteins or fragments thereof, have led to thewidely accepted idea that the “inhibitory region”(residues 96-116) of Tnl binds to an a-helical segment of TnC comprising residues 89-100 in the nonregulatory, COOH-terminal domain. In an attempt to identify other possible physiologically important interactions between these proteins, 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) was used toproduce zero-length cross-links in the complex of rabbit skeletal muscle TnC and Tnl. TnC was activated with EDC and iV-hydroxysuccinimide (NHS) and then mixed with an equimolar amount of Tnl [Grabarek, Z., & Gergely, J.(1988) Biophys. J. 53, 392a]. The resulting cross-linked TnCXI was cleaved with cyanogen bromide, trypsin, and Staphylococcus aureus V8 protease (SAP). Cross-linkedpeptides were purified by reverse-phase HPLC and characterized by sequence analysis. The results indicated that residues from the regulatory Ca2+-bindingsite II in the NH2-terminal domain of TnC (residues 46-78) formed cross-links with Tnl segments spanning residues 92-167. Themost highly cross-linked residues in Tnl were Lys-105 and Lys-107, located in the inhibitory region. These results yield the first evidence for an interaction between the N-terminal domain of TnC and the inhibitory region of Tnl. e Ca2+-dependent change in interaction between the Ca2+-binding (TnC) 1 and the inhibitory (Tnl) components of troponin is one of the key events in the process of activation of contraction in skeletal muscle [for reviews, see Leavis and Gergely (1984) and Zot and Potter (1987)]. In order to understand the molecular basis of muscular activation, much effort has been made to characterize the TnC-Tnl interface. The amino acid sequences of rabbit fast skeletal muscle TnC (Collins et al., 1973, 1977) and Tnl (Wilkinson & Grand, 1975) are known, and these proteins haveserved as models for extensive structure-function studies carried out in several laboratories. While much remains to be learned about the three-dimensional structure of Tnl, analysis of the TnC se-quence (Collins et al., 1973) predicted the locations of four Ca2+-binding sites, designated I-IV going from the amino to the carboxyl terminus of the protein. This prediction was later confirmed by the crystal structures of chicken (Sundaralingam et al., 1985) and turkey (Herzberg & James, 1985) TnCs,