Laboratory of Structural Biology Research, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Structure. 2010 May 12;18(5):553-62. doi: 10.1016/j.str.2010.02.016.
The ClpA chaperone combines with the ClpP peptidase to perform targeted proteolysis in the bacterial cytoplasm. ClpA monomer has an N-terminal substrate-binding domain and two AAA+ ATPase domains (D1 and D2). ClpA hexamers stack axially on ClpP heptamers to form the symmetry-mismatched protease. We used cryo-electron microscopy to visualize the ClpA-ATPgammaS hexamer, in the context of ClpAP complexes. Two segments lining the axial channel show anomalously low density, indicating that these motifs, which have been implicated in substrate translocation, are mobile. We infer that ATP hydrolysis is accompanied by substantial structural changes in the D2 but not the D1 tier. The entire N domain is rendered invisible by large-scale fluctuations. When deletions of 10 and 15 residues were introduced into the linker, N domain mobility was reduced but not eliminated and changes were observed in enzymatic activities. Based on these observations, we present a pseudo-atomic model of ClpAP holoenzyme, a dynamic proteolytic nanomachine.
ClpA 伴侣与 ClpP 肽酶结合，在细菌细胞质中进行靶向蛋白水解。ClpA 单体具有 N 端底物结合结构域和两个 AAA+ATP 酶结构域（D1 和 D2）。ClpA 六聚体在 ClpP 七聚体上轴向堆叠，形成对称性不匹配的蛋白酶。我们使用冷冻电镜观察 ClpA-ATPγS 六聚体在 ClpAP 复合物中的情况。沿轴向通道的两个片段显示出异常低的密度，表明这些参与底物易位的基序是可移动的。我们推断 ATP 水解伴随着 D2 但不是 D1 层的结构发生重大变化。整个 N 结构域由于大规模波动而变得不可见。当在接头中引入 10 个和 15 个残基的缺失时，N 结构域的流动性降低但没有完全消除，并且观察到酶活性的变化。基于这些观察结果，我们提出了 ClpAP 全酶的拟原子模型，这是一种动态的蛋白水解纳米机器。
