Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Proteins. 2012 Dec;80(12):2758-68. doi: 10.1002/prot.24159. Epub 2012 Sep 15.
ClpB reactivates aggregated proteins in cooperation with DnaK/J. The ClpB monomer contains two nucleotide-binding domains (D1, D2), a coiled-coil domain, and an N-terminal domain attached to D1 with a 17-residue-long unstructured linker containing a Gly-Gly motif. The ClpB-mediated protein disaggregation is linked to translocation of substrates through the central channel in the hexameric ClpB, but the events preceding the translocation are poorly understood. The N-terminal domains form a ring surrounding the entrance to the channel and contribute to the aggregate binding. It was suggested that the N-terminal domain's mobility that is maintained by the unstructured linker might control the efficiency of aggregate reactivation. We produced seven variants of ClpB with modified sequence of the N-terminal linker. To increase the linker's conformational flexibility, we inserted up to four Gly next to the GG motif. To decrease the linker's flexibility, we deleted the GG motif and converted it into GP and PP. We found that none of the linker modifications inhibited the basal ClpB ATPase activity or its capability to form oligomers. However, the modified linker ClpB variants showed lower reactivation rates for aggregated glucose-6-phosphate dehydrogenase and firefly luciferase and a lower aggregate-binding efficiency than wt ClpB. We conclude that the linker does not merely connect the N-terminal domain, but it supports the chaperone activity of ClpB by contributing to the efficiency of aggregate binding and disaggregation. Moreover, our results suggest that selective pressure on the linker sequence may be crucial for maintaining the optimal efficiency of aggregate reactivation by ClpB.
ClpB 与 DnaK/J 合作重新激活聚集的蛋白质。ClpB 单体包含两个核苷酸结合结构域（D1、D2）、一个卷曲螺旋结构域和一个附着在 D1 上的 N 端结构域，N 端结构域与 D1 之间有一个 17 个残基长的无规则连接区，其中包含一个 Gly-Gly 基序。ClpB 介导的蛋白质解聚与底物通过六聚体 ClpB 中心通道的易位有关，但易位之前的事件知之甚少。N 端结构域形成环绕通道入口的环，并有助于聚集物的结合。有人提出，N 端结构域的灵活性由无规则连接区维持，可能控制聚集物重新激活的效率。我们制备了七种具有修饰的 N 端连接区序列的 ClpB 变体。为了增加连接区的构象灵活性，我们在 GG 基序旁边插入了多达四个 Gly。为了降低连接区的灵活性，我们删除了 GG 基序并将其转换为 GP 和 PP。我们发现，连接区的任何修饰都没有抑制基础 ClpB ATP 酶活性或其形成寡聚物的能力。然而，修饰的连接区 ClpB 变体显示出对聚集的葡萄糖-6-磷酸脱氢酶和萤火虫荧光素的再激活率较低，并且与 wt ClpB 相比，聚集物结合效率较低。我们得出结论，连接区不仅连接 N 端结构域，而且通过有助于聚集物结合和解聚的效率，支持 ClpB 的伴侣活性。此外，我们的结果表明，对连接区序列的选择性压力可能对维持 ClpB 聚集物再激活的最佳效率至关重要。
