Inter-ring rotations of AAA ATPase p97 revealed by electron cryomicroscopy

Heidi O Yeung,Andreas Förster,Hajime Niwa,Ciarán Mckeown,Cecilia Bebeacua,Caroline Ewens,Paul S Freemont,Xiaodong Zhang

doi:10.1098/rsob.130142

Abstract

The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes.

Highlights

ATPase associated with various cellular activities (AAA) proteins are widespread in all types of cells and use the chemical energy released by hydrolysing ATP to perform various biological functions [1]
We show that one of these is highly dynamic throughout the nucleotide hydrolysis cycle: the position of the N domains above or in plane with the D1 ring is linked to different conformations of the D1 and D2 rings
Positioned either coplanar with or in various positions above the D1 ring, the N domains are a major source of p97 conformational heterogeneity irrespective of nucleotide state, and two major p97 conformations can be described based on the position of the N domains

Summary

Introduction

ATPase associated with various cellular activities (AAA) proteins are widespread in all types of cells and use the chemical energy released by hydrolysing ATP to perform various biological functions [1]. One important and ubiquitous member of the AAA family, conserved throughout evolution and essential for cell viability [2], is p97 (Cdc in yeast). In complex with a large number of cofactors, it participates in numerous cellular activities throughout the cell cycle [3]. P97/Cdc has been implicated, among other processes, in membrane fusion [6], endoplasmic reticulum-associated degradation [7], nucleus reformation after mitosis [8] and DNA damage response [9]. The key to the functional diversity of p97 is the binding of specific cofactors that direct p97 down different functional paths [10] within the ubiquitin proteasome system implicating p97 as a major regulator of global protein turnover

Methods

Findings

Discussion

Conclusion