Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

Abstract

The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.

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

“…PafE rings have a low affinity for 20S CPs, but shortening the C-termini of PafE without deleting the GQYL motifs (PafE Δ155-166 ) increases its affinity for 20S CPs by reducing the distance between a PafE ring and a 20S CP α-ring (13). To better understand the interaction between PafE rings and 20S CPs, we used PafE Δ155-166 with 20S CPs for single particle cryo-EM analysis.…”

“…However, the local resolution varied considerably: the 20S CP region was in the resolution range of 4 -6 Å, with resolved secondary structures and even a few large side chains, but the PafE ring had a resolution of ~10 Å. The low resolution of PafE density was expected due to the flexible PafE Ctermini interacting with the 20S CPs (13,16). For the doubly PafE-capped 20S CP, we masked PafE densities at both ends of the CP particle, and refined the 20S CP region alone by applying D7 symmetry, which led to an improved 3D density map at 3.4 Å average resolution (Supplemental Figs.…”

“…2B). We previously demonstrated K52 of PrcA is essential for PafEdependent degradation (13). To test the significance of the interaction between PafE Y173 and PrcA R26, we substituted R26 to an alanine (PrcA R26A ) and tested if the mutated 20S CP could degrade HspR.…”

“…In contrast, the PafEproteasome neither requires ATP nor interaction with a post-translational modification on a doomed protein for degradation (8,11,12). Instead, it appears that PafE can facilitate the degradation of unfolded proteins and at least one native protein, HspR, without a post-translational modification (11)(12)(13). A failure to degrade HspR by the PafEproteasome results in slowed growth in vitro (12) and likely contributes to the attenuated growth phenotype in vivo (11).…”

“…Like PA26 and PA28, PafE also has a four a-helix bundle fold (13)(14)(15). However, PafE is unique in two ways: (1) its unstructured, proteasome-activating C-terminus is 21 amino acids long, which is nearly twice the length of the unstructured C-termini of PA26 and PA28; and (2) PafE monomers assemble into dodecameric rings with 12-fold symmetry (13,16), in contrast to the heptameric rings of PA26 and PA28 (17). We previously investigated the function of the extended PafE C-terminus and found it confers suboptimal binding to 20S CPs; shortening the C-terminus while maintaining the GQYL motif results in a dramatic increase in proteolysis by Mtb 20S CPs (13).…”