Hidden dynamics of human proteasome autoregulation during protein degradation

Abstract

The 2.5-MDa 26S proteasome maintains proteostasis and regulates myriad cellular processes. How polyubiquitylated substrate interactions regulate proteasome activity is not understood. Here we reconstitute hidden dynamics of proteasome autoregulation during protein degradation by cryo-electron microscopy. We identified 64 conformers of the substrate-bound human 26S proteasome via deep classification. These structures unexpectedly revealed substrate-dependent conformational entanglement of two regulatory particles in the doubly capped holoenzymes and energetic differences between doubly and singly capped ones. Novel ubiquitin-binding sites were delineated on the RPN2, RPN10 and α5 subunits, which remodel polyubiquitin chains for deubiquitylation and recycling. Importantly, single-nucleotide-exchange dynamics of proteasomal AAA-ATPase motor were choregraphed during translocation initiation, which upregulates proteolytic activity by allosterically promoting nucleophilic attack. Our systematic analysis illuminates a grand hierarchical allostery for proteasome autoregulation.