An empirical energy landscape reveals mechanism of proteasome in polypeptide translocation.

Rui Fang,Mengying Zhou,Ying Lu,Jason Hon

doi:10.7554/elife.71911

Abstract

The ring-like ATPase complexes in the AAA+ family perform diverse cellular functions that require coordination between the conformational transitions of their individual ATPase subunits (Erzberger and Berger, 2006; Puchades et al., 2020). How the energy from ATP hydrolysis is captured to perform mechanical work by these coordinated movements is unknown. In this study, we developed a novel approach for delineating the nucleotide-dependent free-energy landscape (FEL) of the proteasome's heterohexameric ATPase complex based on complementary structural and kinetic measurements. We used the FEL to simulate the dynamics of the proteasome and quantitatively evaluated the predicted structural and kinetic properties. The FEL model predictions are consistent with a wide range of experimental observations in this and previous studies and suggested novel mechanistic features of the proteasomal ATPases. We find that the cooperative movements of the ATPase subunits result from the design of the ATPase hexamer entailing a unique free-energy minimum for each nucleotide-binding status. ATP hydrolysis dictates the direction of substrate translocation by triggering an energy-dissipating conformational transition of the ATPase complex.

Highlights

The ring-shaped oligomeric ATPases control key biological processes including protein folding, transcription, DNA replication, cellular cargo transport and protein turnover[1,2,3]
A description of the free-energy landscape (FEL) of proteasome is represented by the potential of mean force of a specific proteasome population measured as a 109 bivariate function of its conformational coordinates and the nucleotide distribution[17,18]
We exercised the principle of parsimony to reconstruct the FEL of the proteasomal ATPase complex and experimentally determined its parameters in an attempt to uncover the mechanism in polypeptide translocation

Summary

Introduction

The ring-shaped oligomeric ATPases control key biological processes including protein folding, transcription, DNA replication, cellular cargo transport and protein turnover[1,2,3]. ATPases assembled from six distinct gene products (RPT1-RPT6) that share 85% sequence identity These ATPases use the energy from ATP hydrolysis to mechanically unfold substrates and translocate them into the CP for proteolysis (Fig. 1A). An unfolded substrate primarily interacts with aromatic residues on the pore-1 loop (PL1) of each ATPase These short structured loops form a righthanded helical “staircase” delineating the interior of the translocation channel. To account for substrate translocation, we and others have proposed that when certain PL1s disengage and move to the top, substrate-engaged PL1s may move in the opposite direction, towards the CP This conformational rearrangement may provide the power stroke to promote axial translocation of substrate (Fig. 1B)[7,8]

Methods

Results

Conclusion