Department of Biochemistry, University of Utah, Salt Lake City, UT, USA.
Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA.
Nat Commun. 2024 Aug 29;15(1):7505. doi: 10.1038/s41467-024-51835-3.
The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase motor domains, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of native structures of Cdc48 affinity purified from budding yeast lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data uncover elements of Shp1-Cdc48 interactions and support a 'hand-over-hand' mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by the D2 motor in substrate translocation/unfolding.
Cdc48 AAA+ ATP 酶是一种丰富且必需的酶，可在多种蛋白质质量控制途径中展开底物。该酶包含两个保守的 AAA+ ATP 酶马达结构域 D1 和 D2，它们组装成具有 D1 堆叠在 D2 上方的六聚体环。在这里，我们报告了从芽殖酵母裂解物中亲和纯化的 Cdc48 与在展开底物过程中充当衔接蛋白 Shp1 的复合物的一组天然结构。我们的分析揭示了跨越整个易位循环的结构快照连续体。这些数据揭示了 Shp1-Cdc48 相互作用的要素，并支持“逐个传递”的机制，其中各个亚基的顺序运动密切协调。D1 在 D2 之前水解 ATP 并与底物脱离，而 D2 在 D1 之前重新结合 ATP 并与底物重新结合，从而解释了 D2 马达在底物易位/展开中起主导作用的原因。
