Nakazaki Yosuke, Watanabe Yo-Hei
Department of Biology, Faculty of Science and Engineering, Konan University, Okamoto 8-9-1, Kobe, 658-8501, Japan; Institute for Integrative Neurobiology, Konan University, Okamoto 8-9-1, Kobe, 658-8501, Japan.
Genes Cells. 2014 Dec;19(12):891-900. doi: 10.1111/gtc.12188. Epub 2014 Oct 7.
ClpB disaggregase forms a ring-shaped hexamer that threads substrate proteins through the central pore using energy from ATP. The ClpB protomer consists of an N-terminal domain, a middle domain, and two AAA+ modules. These two AAA+ modules bind and hydrolyze ATP and construct the core of the hexameric ring. Here, we investigated the roles of the two AAA+ modules in substrate threading. BAP is an engineered ClpB that can bind ClpP proteolytic chamber; substrates threaded by BAP are degraded by ClpP. We combined BAP with conserved motif mutations in two AAA+ modules and measured the steady-state rates of threading of soluble denatured proteins by these mutants over a range of substrate concentrations. By fitting the data to the Michaelis-Menten equation, k(cat) and K(m) values were determined. We found that the kinetic parameters of the substrate threading correlate with the type of mutation introduced rather than the ATPase activity of the mutant. Moreover, some mutants having no or marginal ATPase activity could thread denatured proteins significantly. These results indicate that ClpB can passively thread soluble denatured proteins.
ClpB解聚酶形成一个环形六聚体，利用ATP提供的能量将底物蛋白穿过中心孔。ClpB单体由一个N端结构域、一个中间结构域和两个AAA+模块组成。这两个AAA+模块结合并水解ATP，构建六聚体环的核心。在此，我们研究了两个AAA+模块在底物穿入过程中的作用。BAP是一种经过工程改造的ClpB，它可以结合ClpP蛋白水解腔；由BAP穿入的底物会被ClpP降解。我们将BAP与两个AAA+模块中的保守基序突变相结合，并在一系列底物浓度下测量了这些突变体对可溶性变性蛋白的稳态穿入速率。通过将数据拟合到米氏方程，确定了k(cat)和K(m)值。我们发现底物穿入的动力学参数与引入的突变类型相关，而不是与突变体的ATP酶活性相关。此外，一些没有或只有少量ATP酶活性的突变体也能显著穿入变性蛋白。这些结果表明，ClpB可以被动地穿入可溶性变性蛋白。
