Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne 3086, Victoria, Australia.
Department of Protein Evolution, Max-Planck-Institute for Developmental Biology, D-72076 Tübingen, Germany.
Biomolecules. 2020 Apr 16;10(4):615. doi: 10.3390/biom10040615.
In , SigmaS (σ) is the master regulator of the general stress response. The cellular levels of σ are controlled by transcription, translation and protein stability. The turnover of σ, by the AAA+ protease (ClpXP), is tightly regulated by a dedicated adaptor protein, termed RssB (Regulator of Sigma S protein B)-which is an atypical member of the response regulator (RR) family. Currently however, the molecular mechanism of σ recognition and delivery by RssB is only poorly understood. Here we describe the crystal structures of both RssB domains (RssB and RssB) and the SAXS analysis of full-length RssB (both free and in complex with σ). Together with our biochemical analysis we propose a model for the recognition and delivery of σ by this essential adaptor protein. Similar to most bacterial RRs, the N-terminal domain of RssB (RssB) comprises a typical mixed (βα)-fold. Although phosphorylation of RssB (at Asp58) is essential for high affinity binding of σ, much of the direct binding to σ occurs via the C-terminal effector domain of RssB (RssB). In contrast to most RRs the effector domain of RssB forms a β-sandwich fold composed of two sheets surrounded by α-helical protrusions and as such, shares structural homology with serine/threonine phosphatases that exhibit a PPM/PP2C fold. Our biochemical data demonstrate that this domain plays a key role in both substrate interaction and docking to the zinc binding domain (ZBD) of ClpX. We propose that RssB docking to the ZBD of ClpX overlaps with the docking site of another regulator of RssB, the anti-adaptor IraD. Hence, we speculate that docking to ClpX may trigger release of its substrate through activation of a "closed" state (as seen in the RssB-IraD complex), thereby coupling adaptor docking (to ClpX) with substrate release. This competitive docking to RssB would prevent futile interaction of ClpX with the IraD-RssB complex (which lacks a substrate). Finally, substrate recognition by RssB appears to be regulated by a key residue (Arg117) within the α5 helix of the N-terminal domain. Importantly, this residue is not directly involved in σ interaction, as σ binding to the R117A mutant can be restored by phosphorylation. Likewise, R117A retains the ability to interact with and activate ClpX for degradation of σ, both in the presence and absence of acetyl phosphate. Therefore, we propose that this region of RssB (the α5 helix) plays a critical role in driving interaction with σ at a distal site.
在 中，SigmaS（σ）是一般应激反应的主要调节因子。σ的细胞水平受转录、翻译和蛋白质稳定性的控制。由 AAA+蛋白酶（ClpXP）进行的σ周转受到专门的衔接蛋白 RssB（Sigma S 蛋白 B 的调节剂）的严格调节-它是响应调节剂（RR）家族的非典型成员。然而，目前 RssB 识别和输送 σ 的分子机制理解得还很差。在这里，我们描述了 RssB 两个结构域（RssB 和 RssB）的晶体结构以及全长 RssB 的 SAXS 分析（无论是游离的还是与 σ 复合的）。结合我们的生化分析，我们提出了一个模型，用于描述这个重要衔接蛋白对 σ 的识别和输送。与大多数细菌 RR 一样，RssB 的 N 端结构域（RssB）包含一个典型的混合（βα）折叠。尽管 RssB（在天冬氨酸 58 位）的磷酸化对于 σ 的高亲和力结合是必不可少的，但与 σ 的直接结合主要通过 RssB 的 C 端效应结构域（RssB）进行。与大多数 RR 不同，RssB 的效应结构域形成由两个薄片组成的β-三明治折叠，由α-螺旋突出物环绕，因此与表现出 PPM/PP2C 折叠的丝氨酸/苏氨酸磷酸酶具有结构同源性。我们的生化数据表明，该结构域在底物相互作用和与 ClpX 的锌结合结构域（ZBD）对接中都起着关键作用。我们提出，RssB 与 ClpX 的对接与另一个 RssB 调节剂 IraD 的对接重叠。因此，我们推测与 ClpX 的对接可能通过激活“关闭”状态（如在 RssB-IraD 复合物中看到的）触发其底物的释放，从而将衔接蛋白的对接（与 ClpX）与底物的释放偶联起来。这种竞争性对接 RssB 可能会阻止 ClpX 与 IraD-RssB 复合物（缺乏底物）的无效相互作用。最后，RssB 对底物的识别似乎受到 N 端结构域α5 螺旋中关键残基（精氨酸 117）的调节。重要的是，该残基不直接参与 σ 相互作用，因为 σ 与 R117A 突变体的结合可以通过磷酸化来恢复。同样，R117A 仍然能够与 ClpX 相互作用并激活其降解 σ，无论是在存在还是不存在乙酰磷酸的情况下。因此，我们提出 RssB 的这个区域（α5 螺旋）在远距离与 σ 相互作用中起着关键作用。
