PDF(Sci-hub)
Abstract：
The ATP-dependent degradation of polyubiquitylated proteins by the 26S proteasome is essential for the maintenance of proteome stability and the regulation of a plethora of cellular processes. Degradation of substrates is preceded by the removal of polyubiquitin moieties through the isopeptidase activity of the subunit Rpn11. Here we describe three crystal structures of the heterodimer of the Mpr1-Pad1-N-terminal domains of Rpn8 and Rpn11, crystallized as a fusion protein in complex with a nanobody. This fusion protein exhibits modest deubiquitylation activity toward a model substrate. Full activation requires incorporation of Rpn11 into the 26S proteasome and is dependent on ATP hydrolysis, suggesting that substrate processing and polyubiquitin removal are coupled. Based on our structures, we propose that premature activation is prevented by the combined effects of low intrinsic ubiquitin affinity, an insertion segment acting as a physical barrier across the substrate access channel, and a conformationally unstable catalytic loop in Rpn11. The docking of the structure into the proteasome EM density revealed contacts of Rpn11 with ATPase subunits, which likely stabilize the active conformation and boost the affinity for the proximal ubiquitin moiety. The narrow space around the Rpn11 active site at the entrance to the ATPase ring pore is likely to prevent erroneous deubiquitylation of folded proteins.
摘要：
26S蛋白酶体对多泛素化蛋白的ATP依赖性降解对于维持蛋白质组稳定性和调节过多的细胞过程至关重要。在底物降解之前,通过亚基Rpn11的异肽酶活性除去聚泛素部分。在这里，我们描述了Rpn8和Rpn11的Mpr1-Pad1-N末端结构域的异二聚体的三个晶体结构，结晶为与纳米抗体复合的融合蛋白。该融合蛋白对模型底物表现出适度的去泛素化活性。完全激活需要将Rpn11掺入26S蛋白酶体，并且依赖于ATP水解，表明底物处理和聚泛素去除是耦合的。根据我们的结构，我们建议通过低内在泛素亲和力的联合作用来防止过早激活，充当跨衬底接入通道的物理屏障的插入段，和Rpn11中构象不稳定的催化回路。该结构与蛋白酶体EM密度的对接揭示了Rpn11与ATPase亚基的接触，这可能稳定活性构象并增强对近端泛素部分的亲和力。在ATPase环孔入口处的Rpn11活性位点周围的狭窄空间可能防止折叠蛋白质的错误去泛素化。
