Atypical AAA+ Subunit Packing Creates an Expanded Cavity for Disaggregation by the Protein-Remodeling Factor Hsp104

Petra Wendler,James Shorter,Celia Plisson,Anil G Cashikar,Susan Lindquist,Helen R Saibil

doi:10.1016/j.cell.2007.10.047

Abstract

SummaryHsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe stress by disaggregating denatured proteins through a poorly understood mechanism. Here, we present cryo-electron microscopy maps and domain fitting of Hsp104 hexamers, revealing an unusual arrangement of AAA+ modules with the prominent coiled-coil domain intercalated between the AAA+ domains. This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains. The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction. The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini.

Highlights

The 104 kDa protein-remodeling factor Hsp104 from S. cerevisiae disaggregates chemically or thermally denatured proteins in an ATP dependent manner, and cooperates with the Hsp70/Hsp40 chaperone system to refold the proteins to their native state (Glover and Lindquist, 1998)
Cryo-EM Maps of DN and Full-Length Hsp104 Hexamers Show an Ordered Ring of N Domains Capping a Large Chamber Cryo-EM studies on Hsp100 proteins have so far not revealed any clear density for the N-terminal domains, suggesting that these domains are highly mobile in Hsp100 proteins and undergo movements of at least 30 A (Lee et al, 2003; Ishikawa et al, 2004)
The sensor-1 mutation in NBD2 has no effect on nucleotide binding but reduces ATP hydrolysis by Hsp104 at low protein and ATP concentrations, and elicits several protein-remodeling activities (Hattendorf and Lindquist, 2002b; Doyle et al, 2007)

Summary

Introduction

The 104 kDa protein-remodeling factor Hsp104 from S. cerevisiae disaggregates chemically or thermally denatured proteins in an ATP dependent manner, and cooperates with the Hsp70/Hsp chaperone system to refold the proteins to their native state (Glover and Lindquist, 1998). Class Clp/Hsp100 proteins contain variable N- and C-terminal domains and two highly conserved AAA+ nucleotide binding domains (NBD1 and NBD2) separated by a variable middle region. X-ray crystal structures of subunits of the full-length, bacterial Clp/Hsp100 proteins ClpA (Guo et al, 2002) and ClpB (Lee et al, 2003) show that the two AAA+ domains are stacked head-totail. The ClpB monomers do not form a hexamer, but rather assemble into a spiral containing three ClpB molecules in different conformations Superposition of these structures indicates that the individual domains move as rigid bodies around hinge regions enabling a high mobility, in particular for the coiledcoil and N-terminal domains (Lee et al, 2003). Fitting of the ClpB crystal structure into cryo-EM reconstructions led to a hexameric model in which the coiled-coil domain protrudes from NBD1 on the outside of the complex (Lee et al, 2003, 2007)

Results

Discussion

Conclusion