Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase.

Kamila B Franke,Bernd Bukau,Axel Mogk

doi:10.3389/fmolb.2017.00006

Kamila B Franke, Bernd Bukau + Show 1 more

Open Access

https://doi.org/10.3389/fmolb.2017.00006

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Abstract

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.

Highlights

AAA+ proteins constitute a protein superfamily sharing the ability to convert the chemical energy derived from ATP hydrolysis into mechanical work
We selected the conserved A328 residue for analysis as it is located in a strategic position at the AAA-1 subunit interfaces close to the essential arginine fingers R331 and R332
We show that A328, like the arginine fingers, acts in both, intra-ring and interring signaling

Summary

Introduction

AAA+ proteins constitute a protein superfamily sharing the ability to convert the chemical energy derived from ATP hydrolysis into mechanical work. AAA+ proteins share the AAA domain, which is defined by a region of ∼230 amino acids in length, comprising conserved Walker A and Walker B motifs for nucleotide binding and hydrolysis. The AAA domain drives protein oligomerization, frequently into hexameric ring-like structures with a central pore. Substrate unfolding by AAA+ proteins is typically mediated by pulling at a bound substrate stretch leading to substrate threading through the central pore. This threading activity is executed by pore-located aromatic residues that are located on mobile loops, which move downwards the central channel in a nucleotide-controlled manner (Yamada-Inagawa et al, 2003; Schlieker et al, 2004; Zolkiewski, 2006)

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