Cysteine 81 Is Critical for the Interaction of S100A4 and Myosin-IIA
Cysteine 81 Is Critical for the Interaction of S100A4 and Myosin-IIA
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DOI：
10.1021/bi200853y
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发表时间：
2011-08-23
期刊：
影响因子：
2.9
通讯作者：
Bresnick, Anne R.
中科院分区：
文献类型：
--
作者：
Dulyaninova, Natalya G.;Hite, Karen M.;Bresnick, Anne R.
S100A4 是 Ca2+ 结合蛋白 S100 家族的成员，其过度表达与许多人类病理学相关，包括纤维化、炎症性疾病和转移性疾病。破坏 S100A4/靶点相互作用的小分子的鉴定提供了抑制 S100A4 介导的细胞活动及其相关病理的机制。使用各向异性测定法监测肌球蛋白-IIA 与 S100A4 的 Ca2+ 依赖性结合，NSC 95397 被鉴定为一种抑制剂，可破坏 S100A4/肌球蛋白-IIA 相互作用并抑制 S100A4 介导的肌球蛋白-ILA 丝解聚。质谱分析表明 NSC 95397 与 Cys81 和 Cys86 形成共价加合物，这些加合物位于典型的靶标结合间隙中。诱变研究表明，仅 Cys81 的共价修饰就足以抑制 S100A4 与肌球蛋白-IIA 结合和解聚相关的功能。值得注意的是，用丝氨酸或丙氨酸取代 Cys81 显着损害了 S100A4 促进肌球蛋白-IIA 丝分解的能力。由于已在几种 S100 蛋白中观察到可逆共价半胱氨酸修饰，我们提出 Cys81 的修饰可能为介导 S100A4 与肌球蛋白-IIA 的结合提供额外的调节机制。
Overexpression of S100A4, a member of the S100 family of Ca2+-binding proteins, is associated with a number of human pathologies, including fibrosis, inflammatory disorders, and metastatic disease. The identification of small molecules that disrupt S100A4/target interactions provides a mechanism for inhibiting S100A4-mediated cellular activities and their associated pathologies. Using an anisotropy assay that monitors the Ca2+-dependent binding of myosin-IIA to S100A4, NSC 95397 was identified as an inhibitor that disrupts the S100A4/myosin-IIA interaction and inhibits S100A4-mediated depolymerization of myosin-ILA filaments. Mass spectrometry demonstrated that NSC 95397 forms covalent adducts with Cys81 and Cys86, which are located in the canonical target binding cleft. Mutagenesis studies showed that covalent modification of just Cys81 is sufficient to inhibit S100A4 function with respect to myosin-IIA binding and depolymerization. Remarkably, substitution of Cys81 with serine or alanine significantly impaired the ability of S100A4 to promote myosin-IIA filament disassembly. As reversible covalent cysteine modifications have been observed for several S100 proteins, we propose that modification of Cys81 may provide an additional regulatory mechanism for mediating the binding of S100A4 to myosin-IIA.