Long-range allosteric regulation of the human 26S proteasome by 20S proteasome-targeting cancer drugs

Ashwin Chari,Felix Lambrecht,Jil Schrader,David Haselbach,Fabian Henneberg,Holger Stark

doi:10.1038/ncomms15578

Abstract

The proteasome holoenzyme is the major non-lysosomal protease; its proteolytic activity is essential for cellular homeostasis. Thus, it is an attractive target for the development of chemotherapeutics. While the structural basis of core particle (CP) inhibitors is largely understood, their structural impact on the proteasome holoenzyme remains entirely elusive. Here, we determined the structure of the 26S proteasome with and without the inhibitor Oprozomib. Drug binding modifies the energy landscape of conformational motion in the proteasome regulatory particle (RP). Structurally, the energy barrier created by Oprozomib triggers a long-range allosteric regulation, resulting in the stabilization of a non-productive state. Thereby, the chemical drug-binding signal is converted, propagated and amplified into structural changes over a distance of more than 150 Å from the proteolytic site to the ubiquitin receptor Rpn10. The direct visualization of changes in conformational dynamics upon drug binding allows new ways to screen and develop future allosteric proteasome inhibitors.

Highlights

The proteasome holoenzyme is the major non-lysosomal protease; its proteolytic activity is essential for cellular homeostasis
We determined the high-resolution structures of the human 26S proteasome holoenzyme bound to the chemotherapeutic Oprozomib and its apo form using single particle cryo-electron microscopy and determined the dynamic properties of the proteasome
To address the question how 20S inhibitors affect the proteasome holoenzyme structure, we initially considered the conformational motions of the regulatory particle (RP), which have been described previously (Fig. 1g)[4,6,7]

Summary

Introduction

The proteasome holoenzyme is the major non-lysosomal protease; its proteolytic activity is essential for cellular homeostasis. The Oprozomib–26S proteasome structure (Supplementary Tables 1 and 2) exhibits well-defined densities for the entire proteasome holoenzyme (with the exception of Rpn[1], Supplementary Fig. 4), showing numerous amino-acid side chains in most parts of the molecule and relatively small variations in local resolution (Fig. 1c). Regions encompassing both b subunit rings and the a subunit ring of the CP bound to the RP, as well as the ATPase of the RP are resolved at a resolution range of 3.5–4.5 Å. The B-factors of the model correlate well with the local resolution differences visible in the EM density map (Fig. 1c,d)

Methods

Results

Conclusion