Koike-Takeshita Ayumi, Mitsuoka Kaoru, Taguchi Hideki
From the Department of Applied Bioscience, Kanagawa Institute of Technology, 1030 Shimo-ogino, Atsugi, Kanagawa 243-0292, Japan.
Technology Research Association for Next Generation Natural Products Chemistry, AIST Tokyo Waterfront, 2-3-26, Aomi, Koto-ku, Tokyo 135-0064, Japan, and.
J Biol Chem. 2014 Oct 24;289(43):30005-11. doi: 10.1074/jbc.M114.593822. Epub 2014 Sep 8.
The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was ∼ 20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of ∼ 150 h (∼ 6 days), providing a good model to characterize the football-shaped complex.
大肠杆菌伴侣蛋白GroEL是一种双环伴侣蛋白，它借助GroES和ATP协助蛋白质折叠。GroEL中的天冬氨酸398（Asp-398）是ATP水解的关键残基之一，因为GroEL（D398A）突变体在ATP水解方面存在缺陷（<野生型的2%），但在ATP结合方面没有缺陷。在古菌II组伴侣蛋白中，除了Asp-398外，大肠杆菌GroEL中对应的另一个天冬氨酸残基Asp-52对ATP水解也很重要。我们研究了Asp-52在GroEL中的作用，发现GroEL（D52A）和GroEL（D52A/D398A）突变体的ATP酶活性分别约为野生型GroEL的20%和<0.01%，这表明大肠杆菌GroEL中的Asp-52也参与ATP水解。在ATP存在的情况下，GroEL（D52A/D398A）形成了对称的足球形GroEL-GroES复合物，再次证实了对称复合物在GroEL ATP酶循环中的重要性。值得注意的是，GroEL（D52A/D398A）的对称复合物极其稳定，半衰期约为150小时（约6天），为表征足球形复合物提供了一个良好的模型。
