Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York, USA.
Laboratory of Cellular Biophysics, The Rockefeller University, New York, New York, USA.
J Biol Chem. 2022 Oct;298(10):102457. doi: 10.1016/j.jbc.2022.102457. Epub 2022 Sep 5.
AAA+ (ATPases Associated with diverse cellular Activities) proteases unfold substrate proteins by pulling the substrate polypeptide through a narrow pore. To overcome the barrier to unfolding, substrates may require extended association with the ATPase. Failed unfolding attempts can lead to a slip of grip, which may result in substrate dissociation, but how substrate sequence affects slippage is unresolved. Here, we measured single molecule dwell time using total internal reflection fluorescence microscopy, scoring time-dependent dissociation of engaged substrates from bacterial AAA+ ATPase unfoldase/translocase ClpX. Substrates comprising a stable domain resistant to unfolding and a C-terminal unstructured tail, tagged with a degron for initiating translocase insertion, were used to determine dwell time in relation to tail length and composition. We found greater tail length promoted substrate retention during futile unfolding. Additionally, we tested two tail compositions known to frustrate unfolding. A poly-glycine tract (polyG) promoted release, but only when adjacent to the folded domain, whereas glycine-alanine repeats (GAr) did not promote release. A high complexity motif containing polar and charged residues also promoted release. We further investigated the impact of these and related motifs on substrate degradation rates and ATP consumption, using the unfoldase-protease complex ClpXP. Here, substrate domain stability modulates the effects of substrate tail sequences. polyG and GAr are both inhibitory for unfolding, but act in different ways. GAr motifs only negatively affected degradation of highly stable substrates, which is accompanied by reduced ClpXP ATPase activity. Together, our results specify substrate characteristics that affect unfolding and degradation by ClpXP.
AAA+（与多种细胞活动相关的 ATP 酶）蛋白酶通过将底物多肽穿过狭窄的孔来展开底物蛋白。为了克服展开的障碍，底物可能需要与 ATP 酶延长结合。失败的展开尝试可能导致握持打滑，这可能导致底物解离，但底物序列如何影响打滑仍未解决。在这里，我们使用全内反射荧光显微镜测量了单分子停留时间，对从细菌 AAA+ATP 酶解旋酶/移位酶 ClpX 上结合的底物的时间依赖性解离进行评分。使用包含对展开有抗性的稳定结构域和带有降解标签的 C 末端无规卷曲尾巴的底物，以确定尾巴长度和组成与停留时间的关系。我们发现，更长的尾巴会促进在无效展开过程中保留底物。此外，我们测试了两种已知会阻碍展开的尾巴组成。聚甘氨酸（polyG）片段会促进释放，但仅在与折叠结构域相邻时才会促进释放，而甘氨酸-丙氨酸重复序列（GAr）则不会促进释放。含有极性和带电残基的高复杂度基序也会促进释放。我们使用解旋酶-蛋白酶复合物 ClpXP 进一步研究了这些和相关基序对底物降解率和 ATP 消耗的影响。在这里，底物结构域的稳定性调节了底物尾巴序列的影响。polyG 和 GAr 都对展开有抑制作用，但作用方式不同。GAr 基序仅对高度稳定的底物的降解产生负面影响，这伴随着 ClpXP ATP 酶活性的降低。总之，我们的结果指定了影响 ClpXP 展开和降解的底物特征。
