Structural mechanism for nucleotide-driven remodeling of the AAA-ATPase unfoldase in the activated human 26S proteasome

Yanan Zhu,Youdong Mao,Daqi Yu,Ying Lu,Qi Ouyang,Wei Li Wang

doi:10.1038/s41467-018-03785-w

Abstract

The proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all known eukaryotic cells. It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of the substrate–translocation channel leading to the proteolytic chamber of the core particle (CP). Here we report three alternative states of the ATP-γ-S-bound human proteasome, in which the CP gates are asymmetrically open, visualized by cryo-EM at near-atomic resolutions. At least four nucleotides are bound to the AAA-ATPase ring in these open-gate states. Variation in nucleotide binding gives rise to an axial movement of the pore loops narrowing the substrate-translation channel, which exhibit remarkable structural transitions between the spiral-staircase and saddle-shaped-circle topologies. Gate opening in the CP is thus regulated by nucleotide-driven conformational changes of the AAA-ATPase unfoldase. These findings demonstrate an elegant mechanism of allosteric coordination among sub-machines within the human proteasome holoenzyme.

Highlights

The proteasome is a sophisticated adenosine triphosphate (ATP)-dependent molecular machine responsible for protein degradation in all known eukaryotic cells
We collected cryo-electron microscopy (cryo-EM) data of the human proteasome holoenzyme in the presence of 1 mM ATP-γ-S, using a Gatan K2 Summit direct electron detector mounted on a 200-kV cryogenic electron microscope Tecnai Arctica (Fig. 1a and Supplementary Fig. 1)
All previous studies and the present work suggest that full occupancy of six nucleotide-binding sites stabilizes the resting state, in which the core particle (CP) gate is closed in both human and yeast proteasome holoenzymes[13,14,28,29,30]

Summary

Introduction

The proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all known eukaryotic cells It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of the substrate–translocation channel leading to the proteolytic chamber of the core particle (CP). The three structures mainly differ in their AAA-ATPase ring, in which different nucleotide-binding patterns were observed to associate with conformational changes of the base This allows us to visualize remarkable architectural transitions of the pore loops along the substrate–translocation pathway between the spiralstaircase and saddle-shaped-circle topologies. Taken together, these findings demonstrate an elegant mechanism of allosteric coordination among the sub-machines within the proteasome holoenzyme that actively binds nucleotides in preparation for substrate processing

Methods

Results

Conclusion