Abstract
In all domains of life, proteasomes are gated, chambered proteases that require opening by activators to facilitate protein degradation. Twelve proteasome accessory factor E (PafE) monomers assemble into a single dodecameric ring that promotes proteolysis required for the full virulence of the human bacterial pathogen Mycobacterium tuberculosis Whereas the best characterized proteasome activators use ATP to deliver proteins into a proteasome, PafE does not require ATP. Here, to unravel the mechanism of PafE-mediated protein targeting and proteasome activation, we studied the interactions of PafE with native substrates, including a newly identified proteasome substrate, the ParA-like protein, Rv3213c, and with proteasome core particles. We characterized the function of a highly conserved feature in bacterial proteasome activator proteins: a glycine-glutamine-tyrosine-leucine (GQYL) motif at their C termini that is essential for stimulating proteolysis. Using cryo-electron microscopy (cryo-EM), we found that the GQYL motif of PafE interacts with specific residues in the α subunits of the proteasome core particle to trigger gate opening and degradation. Finally, we also found that PafE rings have 40-Å openings lined with hydrophobic residues that form a chamber for capturing substrates before they are degraded, suggesting PafE has a previously unrecognized chaperone activity. In summary, we have identified the interactions between PafE and the proteasome core particle that cause conformational changes leading to the opening of the proteasome gate and have uncovered a mechanism of PafE-mediated substrate degradation. Collectively, our results provide detailed insights into the mechanism of ATP-independent proteasome degradation in bacteria.
Highlights
In all domains of life, proteasomes are gated, chambered proteases that require opening by activators to facilitate protein degradation
Using cryoelectron microscopy, we found that the GQYL motif of proteasome accessory factor E (PafE) interacts with specific residues in the ␣ subunits of the proteasome core particle to trigger gate opening and degradation
Proteasomal degradation in Mycobacterium tuberculosis (Mtb) is promoted by two distinct pathways: an ATP-dependent pathway catalyzed by Mycobacterium proteasomal ATPase (Mpa) and an ATP-independent pathway mediated by PafE [3, 8]
Summary
Mycobacterium tuberculosis; CP, core particle; Pup, prokaryotic ubiquitin-like protein; FSC, Fourier shell correlation; HspR, heat shock protein repressor; ParP, partitioning with the proteasome; PDB, Protein Data Bank. The cross-linking of PafE with 20S CPs may create unintentional interactions between amino acids that are not involved in proteasome gate activation or might obscure relevant structural features [16]. These previous studies were informative, we felt that more details on the mechanism of proteasome gate opening by PafE remained to be elucidated. We performed cryo-EM experiments to show that, the GQYL sequence in PafE makes specific interactions with residues in PrcA to cause a rotation of Mtb 20S CP ␣-subunits This rotation leads to an outward movement and disorder of the PrcA N termini, which opens the gate into the proteasome core protease. We found that the large hydrophobic opening of PafE may give it a previously unrecognized chaperone-like activity with the ability to capture substrates for delivery to 20S CPs for destruction
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