Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome

Abstract

Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the b-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven a-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

“…Our previous crystallographic studies of the Mtb 20S proteasome may provide some clues (39). Compared with the more rigid, symmetrical 20S WT CP, the Mtb 20S OG CP α-rings are more flexible, and are able to rotate by as much as 3.3°and move radially by 1.4 Å to 2.2 Å.…”

“…The full-length and internal deletion mutant PafE proteins were purified as described above. WT 20S, 20S OG , and 20S T1A CPs were purified as described previously (39). In 20S OG CPs, the N-terminal eight residues of the α-subunit were deleted to produce a CP with a constitutively open gate.…”

Proteasome substrate capture and gate opening by the accessory factor PafE from Mycobacterium tuberculosis

“…The expression and purification methods for the fusion protein were the same as those for PafE alone. PafE variants, HspR, and 20S CPs were purified as described previously (5,13,32).…”

Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1–Pba2 chaperone

“…Structural analyses have been reported for precursors of prokaryotic proteasomes, which assemble without dedicated chaperones 31,32 . No structures are available for precursors of the more complex eukaryotic CP bearing the above-mentioned chaperones Ump1 and Pba1-Pba2.…”