Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St Louis, 63130, USA.
Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, USA.
Nat Commun. 2024 Nov 8;15(1):9681. doi: 10.1038/s41467-024-53681-9.
AAA+ proteolytic machines unfold proteins before degrading them. Here, we present cryoEM structures of ClpXP-substrate complexes that reveal a postulated but heretofore unseen intermediate in substrate unfolding/degradation. A ClpX hexamer draws natively folded substrates tightly against its axial channel via interactions with a fused C-terminal degron tail and ClpX-RKH loops that flexibly conform to the globular substrate. The specific ClpX-substrate contacts observed vary depending on the substrate degron and affinity tags, helping to explain ClpXP's ability to unfold/degrade a wide array of different cellular substrates. Some ClpX contacts with native substrates are enabled by upward movement of the seam subunit in the AAA+ spiral, a motion coupled to a rearrangement of contacts between the ClpX unfoldase and ClpP peptidase. Our structures additionally highlight ClpX's ability to translocate a diverse array of substrate topologies, including the co-translocation of two polypeptide chains.
AAA+ 蛋白水解机器在降解蛋白质之前将其展开。在这里，我们展示了 ClpXP-底物复合物的低温电镜结构，揭示了在底物展开/降解过程中一个假设但迄今为止尚未被发现的中间产物。ClpX 六聚体通过与融合的 C 末端降解尾和 ClpX-RKH 环的相互作用，将天然折叠的底物紧紧地拉向其轴向通道，这些环灵活地适应球状底物。观察到的特定 ClpX-底物相互作用因底物降解尾和亲和标签而异，有助于解释 ClpXP 展开/降解广泛不同的细胞底物的能力。一些 ClpX 与天然底物的接触是通过 AAA+ 螺旋中缝亚基的向上运动实现的，这种运动与 ClpX 解旋酶和 ClpP 肽酶之间的接触重新排列相耦合。我们的结构还突出了 ClpX 能够转运多种不同拓扑结构的底物的能力，包括两条多肽链的共转运。
