Motor Mechanism for Protein Threading through Hsp104

Petra Wendler,James Shorter,David Snead,Celia Plisson,Daniel K Clare,Susan Lindquist,Helen R Saibil

doi:10.1016/j.molcel.2009.02.026

Petra Wendler, James Shorter + Show 5 more

Open Access

https://doi.org/10.1016/j.molcel.2009.02.026

Copy DOI

Abstract

SummaryThe protein-remodeling machine Hsp104 dissolves amorphous aggregates as well as ordered amyloid assemblies such as yeast prions. Force generation originates from a tandem AAA+ (ATPases associated with various cellular activities) cassette, but the mechanism and allostery of this action remain to be established. Our cryoelectron microscopy maps of Hsp104 hexamers reveal substantial domain movements upon ATP binding and hydrolysis in the first nucleotide-binding domain (NBD1). Fitting atomic models of Hsp104 domains to the EM density maps plus supporting biochemical measurements show how the domain movements displace sites bearing the substrate-binding tyrosine loops. This provides the structural basis for N- to C-terminal substrate threading through the central cavity, enabling a clockwise handover of substrate in the NBD1 ring and coordinated substrate binding between NBD1 and NBD2. Asymmetric reconstructions of Hsp104 in the presence of ATPγS or ATP support sequential rather than concerted ATP hydrolysis in the NBD1 ring.

Highlights

Members of the AAA+ (ATPases associated with various cellular activities) super family of protein-remodeling factors employ the energy of ATP binding and hydrolysis to dissolve amorphous or amyloid aggregates
In order to understand the conformational changes upon ATP binding and hydrolysis within Hsp104 hexamers, we pursued two cryoelectron microscopy strategies
The reconstructions presented here of Hsp104 hexamers assembled in the presence of ATP and ADP reinforce the notion that the AAA+ domains in Hsp104 form expanded rings enclosing a large central cavity, unlike the classical packing observed in hexameric crystal structures of related proteins (Wendler et al, 2007)

Summary

Introduction

Members of the AAA+ (ATPases associated with various cellular activities) super family of protein-remodeling factors employ the energy of ATP binding and hydrolysis to dissolve amorphous or amyloid aggregates. In the case of adaptor-bound p97 (Beuron et al, 2006) and SV40 large tumor antigen helicase (Gai et al, 2004), a concerted activity of all subunits has been suggested, but some crystal structures of AAA+ proteins and strong evidence from biochemical experiments on ClpX, PAN, and MCM helicase complexes indicate sequential or probabilistic processivity in the superfamily (DeLaBarre and Brunger, 2005; Singleton et al, 2000; Martin et al, 2005; Hersch et al, 2005; Horwitz et al, 2007; Moreau et al, 2007)

Results

Discussion

Conclusion