Mutant Analysis Reveals Allosteric Regulation of CIpB Disaggregase
Mutant Analysis Reveals Allosteric Regulation of CIpB Disaggregase
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DOI：
10.3389/fmolb.2017.00006
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发表时间：
2017-01-01
影响因子：
5
通讯作者：
Mogk, Axel
中科院分区：
文献类型：
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作者：
Franke, Kamila B.;Bukau, Bernd;Mogk, Axel
对E. coli和革兰氏阳性菌S.酿酒酵母ClpB和Hsp 104与Hsp 70伴侣系统在聚集蛋白的溶解中协同作用。聚集体的溶解依赖于ClpB/Hsp 104的底物穿线活性，该活性由两个ATP酶环（AAA-1，AAA-2）中的ATP水解提供动力。ClpB/Hsp 104 ATP酶活性受M-结构域控制，M-结构域与AAA-1环结合以下调ATP水解。通过与Hsp 70结合保持M-结构域从AAA-1环移位，由于增强了原聚体之间的通信，增加了ATP酶活性。这种通讯涉及保守的精氨酸指。控制ClpB/Hsp 104活性是至关重要的，因为具有永久解离的M-结构域的高活性突变体表现出细胞毒性。在此，我们通过对ClpB（A328）的AAA-1结构域亚基界面的保守残基进行突变分析，分析了与M结构域介导的ATP酶调节相关的AAA-1环间通讯。虽然所有A328 X突变体的解聚活性均降低，但它们的ATP酶活性差别很大。ClpB-A328 I/L突变体具有降低的ATP酶活性，并且当与高活性的ClpB-K476 C M-结构域突变组合时，抑制细胞毒性。这强调了通过M结构域解离激活ClpB ATP酶依赖于增加的亚基通讯。相反，ClpB-A328 V突变体具有非常高的ATP酶活性，并且其自身表现出细胞毒性，使其成为新的高活性ClpB突变体。然而，ClpB-A328 V的高活性不同于M-结构域突变体的高活性，因为M-结构域与AAA-1环保持结合。ClpB-A328 V的高ATP酶活性主要依赖于AAA-2环，并与AAA-2催化位点的不同构象变化相关。这些发现表征了亚基界面残基A328作为控制两个AAA环中ATP水解的关键调节元件。
The members of the hexameric AAA+ disaggregase of E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings (AAA-1, AAA-2). ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings.