Abstract
ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically. The results provide insights into nucleotide sensing, explaining how the conserved sensor 2 motif contributes to the discrimination between ADP and ATP binding. Furthermore, the role of a conserved active-site arginine (Arg621), which controls binding of the essential Mg2+ ion, is described. Finally, a hypothesis is presented as to how the ATPase activity is regulated by a conformational switch that involves the essential Walker A lysine. In the proposed model, an unusual side-chain conformation of this highly conserved residue stabilizes a catalytically inactive state, thereby avoiding unnecessary ATP hydrolysis.
Highlights
The molecular chaperone ClpB, the bacterial homologue of Hsp104, is a disaggregation machine that provides thermotolerance by resolubilizing and reactivating aggregated proteins in concert with the DnaK (Hsp70) chaperone system (Sanchez & Lindquist, 1990; Doyle & Wickner, 2009)
ClpB belongs to the family of AAA+ proteins (ATPases associated with various cellular activities) that comprises a wide range of molecular machines which utilize the energy from ATP hydrolysis to remodel their respective substrates (Tucker & Sallai, 2007; Hanson & Whiteheart, 2005)
In order to understand the mechanism behind the force generation upon ATP hydrolysis in the disaggregation machine ClpB, we aimed at obtaining high-resolution crystal structures of one of its two nucleotide-binding domains, nucleotide-binding domain 2 (NBD2), in different nucleotide states
Summary
The molecular chaperone ClpB, the bacterial homologue of Hsp104, is a disaggregation machine that provides thermotolerance by resolubilizing and reactivating aggregated proteins in concert with the DnaK (Hsp70) chaperone system (Sanchez & Lindquist, 1990; Doyle & Wickner, 2009). The nucleotide-binding domains (NBDs) of AAA+ proteins constitute the motor subunits of the molecular machine. They share highly conserved structural features (Wendler et al, 2012). A unifying feature that is shared by all AAA+ Our results provide a better understanding of how the ATPase proteins, but is more generally found amongst various cycle of the molecular motor is regulated and modulated, families of ATPases and GTPases, is the phosphate-binding which is essential in order to gain further insights into the loop (P-loop) or Walker A motif characterized by the functioning of the complete disaggregation machine. AAA+ proteins are active as oligomers, with the catalytic site being located at the interface between two NBD subunits in the oligomer. The sensor 1 motif contains a conserved polar residue (N/T) that is essential
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