Trans-Acting Arginine Residues in the AAA+ Chaperone ClpB Allosterically Regulate the Activity through Inter- and Intradomain Communication

Cathleen Zeymer,Sebastian Fischer,Jochen Reinstein

doi:10.1074/jbc.m114.608828

Cathleen Zeymer, Sebastian Fischer + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m114.608828

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Abstract

The molecular chaperone ClpB/Hsp104, a member of the AAA+ superfamily (ATPases associated with various cellular activities), rescues proteins from the aggregated state in collaboration with the DnaK/Hsp70 chaperone system. ClpB/Hsp104 forms a hexameric, ring-shaped complex that functions as a tightly regulated, ATP-powered molecular disaggregation machine. Highly conserved and essential arginine residues, often called arginine fingers, are located at the subunit interfaces of the complex, which also harbor the catalytic sites. Several AAA+ proteins, including ClpB/Hsp104, possess a pair of such trans-acting arginines in the N-terminal nucleotide binding domain (NBD1), both of which were shown to be crucial for oligomerization and ATPase activity. Here, we present a mechanistic study elucidating the role of this conserved arginine pair. First, we found that the arginines couple nucleotide binding to oligomerization of NBD1, which is essential for the activity. Next, we designed a set of covalently linked, dimeric ClpB NBD1 variants, carrying single subunits deficient in either ATP binding or hydrolysis, to study allosteric regulation and intersubunit communication. Using this well defined environment of site-specifically modified, cross-linked AAA+ domains, we found that the conserved arginine pair mediates the cooperativity of ATP binding and hydrolysis in an allosteric fashion.

Highlights

Two neighboring, trans-acting arginines in the N-terminal AAAϩ domain are essential for oligomerization and activity of ClpB/Hsp104
In agreement with previous experiments on full-length ClpB by Yamasaki et al [17], the single and double mutants R322A, R323A, and R322A/R323A, respectively, showed about 1000fold reduced ATPase activity compared with the wild type, indicating that both conserved arginines are crucial for ATP hydrolysis in ClpB NBD1
We investigated the role of the conserved, transacting arginines Arg-322 and Arg-323 in allosteric regulation and intersubunit communication in the molecular disaggregation machine ClpB (Fig. 5)

Summary

Background

Trans-acting arginines in the N-terminal AAAϩ domain are essential for oligomerization and activity of ClpB/Hsp104. Several AAA؉ proteins, including ClpB/Hsp104, possess a pair of such trans-acting arginines in the N-terminal nucleotide binding domain (NBD1), both of which were shown to be crucial for oligomerization and ATPase activity. There are essential arginine residues, often termed arginine fingers, that contribute to the active sites in trans because they are in close proximity to the nucleotide bound to the adjacent subunit The role of such conserved arginines in AAAϩ proteins has been investigated extensively (16 –19). Inspired by successful work on the mechanism of ClpX, another AAAϩ protein (26 –28), we applied a combined approach of covalently linking NBD1-M subunits and introducing Walker A/B and arginine finger mutations Using these fixed and well determined arrangements of wildtype and mutated subunits in a direct neighborhood, it was possible to dissect the mechanisms of allosteric regulation and intersubunit communication in the AAAϩ chaperone ClpB/Hsp104

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION