Rivera-Rivera Izarys, Román-Hernández Giselle, Sauer Robert T, Baker Tania A
Department of Biology and.
Department of Biology and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139
Proc Natl Acad Sci U S A. 2014 Sep 16;111(37):E3853-9. doi: 10.1073/pnas.1414933111. Epub 2014 Sep 3.
The ClpS adaptor collaborates with the AAA+ ClpAP protease to recognize and degrade N-degron substrates. ClpS binds the substrate N-degron and assembles into a high-affinity ClpS-substrate-ClpA complex, but how the N-degron is transferred from ClpS to the axial pore of the AAA+ ClpA unfoldase to initiate degradation is not known. Here we demonstrate that the unstructured N-terminal extension (NTE) of ClpS enters the ClpA processing pore in the active ternary complex. We establish that ClpS promotes delivery only in cis, as demonstrated by mixing ClpS variants with distinct substrate specificity and either active or inactive NTE truncations. Importantly, we find that ClpA engagement of the ClpS NTE is crucial for ClpS-mediated substrate delivery by using ClpS variants carrying "blocking" elements that prevent the NTE from entering the pore. These results support models in which enzymatic activity of ClpA actively remodels ClpS to promote substrate transfer, and highlight how ATPase/motor activities of AAA+ proteases can be critical for substrate selection as well as protein degradation.
ClpS衔接蛋白与AAA+ ClpAP蛋白酶协同作用，识别并降解N端规则底物。ClpS结合底物N端规则序列并组装成高亲和力的ClpS-底物-ClpA复合物，但N端规则序列如何从ClpS转移至AAA+ ClpA解折叠酶的轴向孔以启动降解尚不清楚。在此，我们证明ClpS的无结构N端延伸（NTE）进入活性三元复合物中的ClpA加工孔。我们证实ClpS仅以顺式促进递送，这通过将具有不同底物特异性以及活性或无活性NTE截短的ClpS变体混合得以证明。重要的是，我们发现通过使用携带阻止NTE进入孔的“阻断”元件的ClpS变体，ClpA与ClpS NTE的结合对于ClpS介导的底物递送至关重要。这些结果支持了ClpA的酶活性积极重塑ClpS以促进底物转移的模型，并突出了AAA+蛋白酶的ATP酶/马达活性对于底物选择以及蛋白质降解的关键作用。
