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

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.

Keywords: Mycobacterium tuberculosis; Proteasome activator; cryo-electron microscopy; proteasome; protein degradation; protein targeting; structural biology.

Figure 1.

Cryo-EM structure of the PafE(Δ155–166)–20S CP complex. A, side view of surface-rendered 3D density map of a PafE(Δ155–166)–20S CP complex, shown as a semi-transparent surface, superimposed with the crystal structure of PafE (salmon, PDB code 5IET) and a refined Mtb 20S CP atomic model (cyan, PDB: 3MI0) in schematics. B, top view of the PafE(Δ155–166)–20S CP density map surface rendered at a lower threshold (5.8σ) showing the weaker density of PafE ring (left) or rendered at the normal threshold (7.0σ) such that most PafE ring is invisible except for the well ordered C termini (right). The densities of the PafE C termini are colored in salmon. The orange and cyan circles mark the circumferences of the PafE and 20S CP, respectively. C, a view of the uncapped end of a 20S CP by 3D density map rendered at the normal threshold (7.0σ). D, left, superimposition of the α-rings of the 20S CP with (in magenta schematic) and without (in cyan schematic) the capping PafE(Δ155–166) ring (not shown), revealing a 3° in-plane rotation of each α-subunit. The PafE GQYL motif is shown as orange sticks. Right: a zoomed view of the area marked by the black box in the left panel. Note that the first modeled residue of the α-subunit is Pro-4 and Ser-8 in the uncapped end and PafE-capped end, respectively.

Figure 2.

Insertion of the PafE GQYL motifs into the 20S CP moves helix 0 (H0) of the α-subunit ring. A, electron density map of the α-ring of the 3.4-Å 3D map of PafE(Δ155–166)-bound 20S CP with D7 symmetry. The PafE C terminus density is colored in salmon. Middle and lower panels are zoomed-in density and a schematic view of interactions between a PafE C terminus (salmon) and 20S CP α-subunits (magenta). The side chains of PafE Tyr-173 and the carbonyl oxygen of PafE Leu-174 interact with side chains of PrcA Arg-26 and PrcA Lys-52, respectively. B, sequence alignment of the proteasomal α-subunits from different organisms: Mtb and Mja, Methanococcus jannaschii; Afu, Archaeoglobus fulgidus; Sce, S. cerevisiae; and Hsa, Homo sapiens. In the cases of eukaryotes (Sce and Hsa), the sequences of α2-subunits were chosen for alignment, because these subunits interact with the activating C termini of the hexameric ATPases Rpt1–6 in the cryo-EM structures of the yeast and human 26S proteasomes (PBD codes 5WVK and 5GJQ). C, SDS-PAGE of in vitro degradation assays using HspR as a substrate. Aliquots were removed at the indicated time points (min) and analyzed by 15% SDS-PAGE. The degradation assays were repeated at least three times with essentially same results. PafE mediates HspR degradation by WT 20S CPs, but not 20S(R26A) or 20S(K52A) CPs. D, an quantification of the proteolysis reactions in C by estimating the relative intensity of each band, showing the amounts of remaining HspR substrate over a 150-min reaction time. E, a zoomed in view of superimposed 20S CP α-rings with (magenta) or without (cyan) a capping PafE ring. PafE residues are labeled in orange, and PrcA residues in black. The black arrows show the movements of the side chains in the two structures.

Figure 3.

PafE rings with six GQYL motifs cannot mediate substrate degradation by Mtb 20S CPs. A, left: diagram of the PafE–PafE protein and an electron micrograph of purified and negative stained PafE–PafE protein. Inset, 2D class averages of PafE-PafE. Right, diagram of wildtype PafE and the electron micrograph and 2D class averages of purified protein at the same scale with that of PafE–PafE. B, SDS-PAGE analysis of proteolysis products at the specified time points. Molecular weight (MW) markers are on the left. The degradation assay was done at least three times with essentially same results.

Figure 4.

ParP co-purifies with PafE and is degraded by Mtb 20S CPs in vitro. A, upper: SDS-PAGE (15% w/v) of ParP–His₆ co-purified with WT PafE (untagged) as a complex, after Ni²⁺-nitrilotriacetate acid column (left panel) or after further purification with a Superose 6 10/300 GL gel filtration column (right panel). Lower, the size exclusion column profile of PafE and PafE–ParP. B, SDS-PAGE (15% w/v) of in vitro proteolysis reaction products. Purified ParP and 20S CPs (both His₆ tagged) were incubated at room temperature with either PafE (upper panel) or PafE(Δ155–174) (lower panel). Aliquots were removed at the indicated time points and analyzed. The degradation assays were done at least three times with essentially same results. C, an quantification of the proteolysis reactions in B by estimating the relative intensity of each band, showing the amounts of remaining ParP substrate over a 150-min reaction time.

Figure 5.

Proteins are targeted for degradation to 20S proteasomes through the central channel of PafE rings. A, representative reference-free 2D class averages of negatively stained PafE alone (upper panel) and ParP–PafE (lower panel) purified from E. coli. Note that some of the PafE alone averages are reuses of the PafE rings shown in Fig. 3A, right panel. B, a cut-open side view of PafE ring showing the interior chamber surface. The surface potential map of PafE (from blue positive charge to red negative charge) is superimposed on the schematic view of the same structure, with the two hydrophobic residues, Pro-65 and Phe-138, lining the chamber surface shown in yellow spheres. C–E, SDS-PAGE (15%) of HspR degradation reaction products. Recombinant 20S CP–His₆ and HspR–His₆ were incubated at room temperature with His₆–PafE (C), PafE(P65G) (D), or PafE_F132E (E). Aliquots were removed at the indicated time points and analyzed. F, quantification of the remaining HspR over the time course by estimating the relative intensities of individual bands. G, a model whereby substrate is held inside a well ordered PafE chamber, and not by the C termini.

Figure 6.

A proposed model for PafE-mediated protein substrate degradation by Mtb 20S CP. The PafE dodecamer has a central channel with a diameter of 40 Å. The sizable chamber is partially hydrophobic, and accommodates small or partially disordered protein substrates for degradation by the Mtb proteasome. The substrate entry gate in the Mtb 20S CP is closed in its free form, and is opened by the C termini of PafE when a PafE ring docks onto the α-ring. The disordered substrate held inside PafE chamber passes through the open gate and reaches the proteolytic site in the β-rings and is subsequently cleaved.