ClpAP, an ATP-dependent protease consisting of ClpA, a double-ring hexameric unfoldase of the ATPases associated with diverse cellular activities superfamily, and the ClpP peptidase, degrades damaged and unneeded proteins to support cellular proteostasis. ClpA recognizes many protein substrates directly, but it can also be regulated by an adapter, ClpS, that modifies ClpA's substrate profile toward N-degron substrates. Conserved tyrosines in the 12 pore-1 loops lining the central channel of the stacked D1 and D2 rings of ClpA are critical for degradation, but the roles of these residues in individual steps during direct or adapter-mediated degradation are poorly understood. Using engineered ClpA hexamers with zero, three, or six pore-1 loop mutations in each ATPases associated with diverse cellular activities superfamily ring, we found that active D1 pore loops initiate productive engagement of substrates, whereas active D2 pore loops are most important for mediating the robust unfolding of stable native substrates. In complex with ClpS, active D1 pore loops are required to form a high affinity ClpA•ClpS•substrate complex, but D2 pore loops are needed to "tug on" and remodel ClpS to transfer the N-degron substrate to ClpA. Overall, we find that the pore-1 loop tyrosines in D1 are critical for direct substrate engagement, whereas ClpS-mediated substrate delivery requires unique contributions from both the D1 and D2 pore loops. In conclusion, our study illustrates how pore loop engagement, substrate capture, and powering of the unfolding/translocation steps are distributed between the two rings of ClpA, illuminating new mechanistic features that may be common to double-ring protein unfolding machines.

中文翻译：

---

D1 和 D2 AAA+ 环的 pore-1 环之间的分工协调 ClpAP 蛋白酶的底物选择性。

ClpAP 是一种 ATP 依赖性蛋白酶，由 ClpA（一种与多种细胞活动超家族相关的 ATP 酶的双环六聚体去折叠酶）和 ClpP 肽酶组成，可降解受损和不需要的蛋白质以支持细胞蛋白稳态。ClpA 直接识别许多蛋白质底物，但它也可以通过适配器 ClpS 进行调节，该适配器将 ClpA 的底物谱修改为 N-degron 底物。ClpA 的堆叠 D1 和 D2 环的中央通道内衬的 12 个 pore-1 环中的保守酪氨酸对降解至关重要，但这些残基在直接或适配器介导的降解过程中的各个步骤中的作用却知之甚少。使用工程化的 ClpA 六聚体，在与不同细胞活动超家族环相关的每个 ATP 酶中具有零个、三个或六个 pore-1 环突变，我们发现活性 D1 孔环启动了底物的有效接合，而活性 D2 孔环对于介导稳定的天然底物的稳健展开最重要。与 ClpS 复合时，需要有活性的 D1 孔环来形成高亲和力的 ClpA•ClpS•底物复合物，但需要 D2 孔环来“拉动”并重塑 ClpS 以将 N-degron 底物转移到 ClpA。总体而言，我们发现 D1 中的 pore-1 环酪氨酸对于直接底物接合至关重要，而 ClpS 介导的底物递送需要 D1 和 D2 孔环的独特贡献。总之，我们的研究说明了孔环接合、底物捕获和展开/易位步骤的动力如何分布在 ClpA 的两个环之间，