Single molecule microscopy reveals diverse actions of substrate sequences that impair ClpX AAA+ ATPase function.
Single molecule microscopy reveals diverse actions of substrate sequences that impair ClpX AAA+ ATPase function.
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DOI：
10.1016/j.jbc.2022.102457
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发表时间：
2022-10
影响因子：
4.8
通讯作者：
Coffino, Philip
中科院分区：
文献类型：
--
作者：
Wang, Xiao;Simon, Sanford M.;Coffino, Philip
AAA+(与不同细胞活动相关的ATPase)蛋白酶通过将底物多肽拉过一个狭窄的孔来展开底物蛋白。为了克服展开的障碍，底物可能需要延长与ATPase的结合。失败的展开尝试可能会导致握力滑移，这可能导致底物解离，但底物顺序如何影响滑脱仍未解决。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，记录了细菌AAA+ATPase展开酶/转位酶ClpX参与底物的随时间变化的解离情况。底物包括抗展开的稳定结构域和C-末端非结构化尾巴，标记有用于启动转位酶插入的降解子，用于确定与尾部长度和组成有关的停留时间。我们发现，在徒劳的展开过程中，较大的尾长促进了底物的保留。此外，我们测试了两种已知会阻碍展开的尾部成分。聚甘氨酸链(PolyG)促进释放，但仅当邻近折叠结构域时，而甘氨酸-丙氨酸重复序列(GAR)不促进释放。含有极性残基和带电残基的高度复杂的基序也促进了释放。我们使用未折叠酶-蛋白酶复合体ClpXP进一步研究了这些基序和相关基序对底物降解率和ATP消耗的影响。在这里，底物结构域稳定性调节底物尾部序列的影响。PolyG和GAR都对去折叠有抑制作用，但作用方式不同。GAR基序只对高稳定底物的降解产生负面影响，并伴随着ClpXP ATPase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性。
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.
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影响因子：
48
作者：
Hua, Boyang;Han, Kyu Young;Zhou, Ruobo;Kim, Hajin;Shi, Xinghua;Abeysirigunawardena, Sanjaya C.;Jain, Ankur;Singh, Digvijay;Aggarwal, Vasudha;Woodson, Sarah A.;Ha, Taekjip
通讯作者：
Ha, Taekjip
DOI：
10.1016/j.bbamcr.2011.06.007
发表时间：
2012-01
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Baker TA;Sauer RT
通讯作者：
Sauer RT
影响因子：
10.5
作者：
Gottesman, S;Roche, E;Sauer, RT
通讯作者：
Sauer, RT
影响因子：
3.8
作者：
FALK, K;GRATAMA, JW;ERNBERG, I
通讯作者：
ERNBERG, I
影响因子：
11.4
作者：
Hoyt, Martin A.;Zich, Judith;Coffino, Philip
通讯作者：
Coffino, Philip