Organization and ligand binding properties of the tail of Acanthamoeba myosin-IA -: Identification of an actin-binding site in the basic (tail homology-1) domain
Organization and ligand binding properties of the tail of Acanthamoeba myosin-IA -: Identification of an actin-binding site in the basic (tail homology-1) domain
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DOI：
10.1074/jbc.274.49.35159
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发表时间：
1999-12-03
影响因子：
4.8
通讯作者：
Pollard, TD
中科院分区：
文献类型：
--
作者：
Lee, WL;Ostap, EM;Pollard, TD
棘阿米巴肌球蛋白-LA 重链基因编码 134 kDa 的蛋白质，具有催化结构域、三个潜在的轻链结合位点以及具有单独折叠尾部同源 (TH) -1、-2 和 -3 结构域的尾部。尽管 TH-1 由 15% 的赖氨酸和精氨酸组成，但对胰蛋白酶消化具有高度抵抗力，TH-2/3 对 α-胰凝乳蛋白酶消化具有抵抗力。 TH-1 和 TH-2/3 之间的肽键可被胰蛋白酶、α-胰凝乳蛋白酶和内切天冬氨酸裂解，但不能被 V8 蛋白酶裂解。TH-2/3 的圆二色光谱显示主要为随机结构、转角和 β 链，但没有 α 螺旋。 21.6 kDa）表明这些结构域与电子显微照片重建中的整个肌球蛋白-I 尾部一样长。此外，单独表达和纯化的TH-1以高亲和力与TH-2/3结合，因此我们提出TH-1和TH-2/3在肌球蛋白-IA尾部并排排列。单独的 TH-1、TH-2 和 TH-2/3 均以高亲和力结合肌肉肌动蛋白丝，盐抑制 TH-2/3 与肌肉肌动蛋白结合，但不抑制阿米巴肌动蛋白丝。 TH-1 在生理盐浓度下增强 TH-2/3 与肌肉肌动蛋白丝的结合，表明 TH-1 和 TH-2/3 在肌动蛋白结合中合作。内在荧光测定表明，TH-2/3 也以高亲和力与蛋白 Acan125 结合，类似于肌球蛋白-IC 的 SH3 结构域，SH3 序列的系统发育分析表明，肌球蛋白-I 在肌球蛋白-I 亚型基因分歧后获得了 SH3 结构域。
The Acanthamoeba myosin-LA heavy chain gene encodes a 134-kDa protein with a catalytic domain, three potential light chain binding sites, and a tail with separately folded tail homology (TH) -1, -2, and -3 domains. TH-1 is highly resistant to trypsin digestion despite consisting of 15% lysine and arginine, TH-2/3 is resistant to alpha-chymotrypsin digestion. The peptide link between TH-1 and TH-2/3 is cleaved by trypsin, alpha-chymotrypsin, and endo-AspN but not V8 protease, The CD spectra of TH-2/3 indicate predominantly random structure, turns, and beta-strands but no alpha-helix, The hydrodynamic properties of TH-2/3 (Stokes' radius of 3.0 nn, sedimentation coefficient of 1.8 S, and molecular mass of 21.6 kDa) indicate that these domains are as long as the whole myosin-I tail in reconstructions of electron micrographs. Furthermore, separately expressed and purified TH-1 binds with high affinity to TH-2/3, Thus we propose that TH-1 and TH-2/3 are arranged side by side in the myosin-IA tail. Separate TH-1, TH-2, and TH-2/3 each binds muscle actin filaments with high affinity, Salt inhibits TH-2/3 binding to muscle actin but not amoeba actin filaments. TH-1 enhances binding of TH-2/3 to muscle actin filaments at physiological salt concentration, indicating that TH-1 and TH-2/3 cooperate in actin binding. An intrinsic fluorescence assay shows that TH-2/3 also binds with high affinity to the protein Acan125 similar to the SH3 domain of myosin-IC, Phylogenetic analysis of SH3 sequences suggests that myosin-I acquired SH3 domain after the divergence of the genes for myosin-I isoforms.