Cryo-EM reveals the conformation of a substrate analogue in the human 20S proteasome core.

Paula C.A Da Fonseca,Edward P Morris

doi:10.1038/ncomms8573

Abstract

The proteasome is a highly regulated protease complex fundamental for cell homeostasis and controlled cell cycle progression. It functions by removing a wide range of specifically tagged proteins, including key cellular regulators. Here we present the structure of the human 20S proteasome core bound to a substrate analogue inhibitor molecule, determined by electron cryo-microscopy (cryo-EM) and single-particle analysis at a resolution of around 3.5 Å. Our map allows the building of protein coordinates as well as defining the location and conformation of the inhibitor at the different active sites. These results open new prospects to tackle the proteasome functional mechanisms. Moreover, they also further demonstrate that cryo-EM is emerging as a realistic approach for general structural studies of protein–ligand interactions.

Highlights

The proteasome is a highly regulated protease complex fundamental for cell homeostasis and controlled cell cycle progression
We explore the use of cryoEM as an emerging tool for the study of protein–ligand interactions, focusing on the structure of the human 20S proteasome core bound to an inhibitor
We determined the structure of the human 20S proteasome core bound to the inhibitor adamantaneacetyl-(6-aminohexanoyl)3(leucyl)3-vinylmethyl-sulfone (AdaAhx3L3VS)[12] by cryo-EM and single-particle analysis

Summary

Introduction

The proteasome is a highly regulated protease complex fundamental for cell homeostasis and controlled cell cycle progression It functions by removing a wide range of tagged proteins, including key cellular regulators. Our map allows the building of protein coordinates as well as defining the location and conformation of the inhibitor at the different active sites These results open new prospects to tackle the proteasome functional mechanisms. The 20S core has limited proteolytic activity against smaller peptides and unfolded proteins[5], and full activation requires the binding of regulatory complexes to the a rings at the ends of the barrel shaped core Such regulatory complexes include the 19S regulatory particles, which bind to the 20S core to form the 26S proteasome and recruit fully folded protein substrates tagged for degradation by ubiquitination[6]. We explore the use of cryoEM as an emerging tool for the study of protein–ligand interactions, focusing on the structure of the human 20S proteasome core bound to an inhibitor

Methods

Results

Conclusion