Structural basis for allosteric regulation of the proteasome core particle

Intracellular protein degradation is vital across all domains of life ¹ . In eukaryotes, the ubiquitin proteasome system performs most non-lysosomal protein degradation and influences numerous cellular processes. Some bacteria, including the human pathogen Mycobacterium tuberculosis ( Mtb ), encode a proteasome system that selectively degrades damaged or misfolded proteins crucial for the pathogen’s survival within host macrophages ^2–7 . Consequently, the 20S core particle (CP), the central component of the proteasome system, has emerged as a viable target for tuberculosis treatment strategies ^2,8–10 . Both eukaryotic and Mtb proteasome systems are allosterically regulated ^11–13 , yet the specific conformations involved have not been captured in high-resolution structures to date. Here we present the first structure of Mtb 20S CP, and indeed any 20S CP, in an inactive state called 20S _OFF , distinguished from the canonical active state, 20S _ON , by the conformation of switch helices I and II. The rearrangement of these helices collapses the S1 pocket, effectively inhibiting substrate binding. The switch helices are conserved and regulate the activity of HslV protease, the proteasome’s ancestral enzyme in bacteria, and a diverse family of serine/threonine protein phosphatases. Our results highlight the potential of harnessing allostery to develop therapeutics against the 20S CP in Mtb and eukaryotic systems.