Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase
| Autor: | Bernd Bukau, Axel Mogk, Kamila B. Franke |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Arginine ATPase Protein subunit Hsp104 Allosteric regulation Mutant macromolecular substances AAA+ protein Biochemistry Genetics and Molecular Biology (miscellaneous) Biochemistry 03 medical and health sciences ATP hydrolysis ClpB Molecular Biosciences protein disaggregation Molecular Biology Original Research biology arginine finger Cell biology 030104 developmental biology Chaperone (protein) biology.protein CLPB |
| Zdroj: | Frontiers in Molecular Biosciences |
| ISSN: | 2296-889X |
| Popis: | 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 AAA1 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. |
| Databáze: | OpenAIRE |
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