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.

Keywords: Mycobacterium tuberculosis; X-ray crystallography; cryo-EM; proteasome; structural biology.

Fig. 1.

PafE forms dodecameric rings. (A) PafE contains four α-helices (cyan cylinders), as well as an additional N-terminal short H0 helix and C-terminal 21-residue peptide, including a terminal GQYL motif (red). In addition to the full-length protein, we also produced two N- and C-terminal truncation constructs PafE_ΔN14ΔC21 and PafE_ΔN43ΔC21. (B) SDS/PAGE analysis of the purified proteins described in A. (C) Crystal structure of the dodecameric PafE_ΔN14ΔC21 in top (Left) and side (Right) views. (D) Surface potential of PafE_ΔN14ΔC21. The positive and negative charges are colored blue and red, respectively.

Fig. 2.

Subunit interface is essential for the integrity of the PafE dodecamer structure. (A) Two neighboring PafE monomers in the dodecameric ring viewed from the top distal surface in rainbow cartoon view. Two concentric arcs are part of the inner and outer shells of the PafE ring. The dashed vertical line marks the subunit interface. Note the close intersubunit H1/H4 packing. (B, Left) Viewed from inside the channel, showing the GXXXGXXXG motif of helix H4 that enables H1/H4 tight packing. The three Gly are shown as spheres. Small or flexible side chains in helix H1 important for the tight packing are indicated. Gln56 and Glu127 form H-bonds. (Right) SEC profiles of WT PafE (blue) in comparison with that of three Gly single-mutation PafE proteins (yellow, PafE_G124I; green, PafE_G128I; and purple, PafE_G132I). (C, Left) Viewed from the surface proximal to 20S CP, residues involved in intersubunit interaction in the outer shell are shown as sticks. Arg99 and Glu84 form two H-bonds, and Arg49 and Glu96 form two H-bonds. (Right) Gel-filtration profiles showed mutagenesis of PafE residues involved in H-bonding affect or abolish native ring formation. Right panels in B and C used the same HiLoad 16/60 Superdex 200-pg column. Size standards are marked between the two chromatographic panels.

Fig. 3.

In vitro binding of PafE to Mtb 20S CPs as measured by isothermal titration calorimetry. The Upper panels show typical raw calorimetry data when PafE was injected at 25 °C into 20S CPs: (A) 480 μM PafE into 33 μM Mtb 20S_WT, and (B and C) 85 μM PafE into 6.7 μM Mtb 20S_OG and 20S_T1A, respectively. Higher protein concentrations were needed to measure the weak binding between PafE and 20S_WT CPs. In the Lower panels, each datapoint represents the reaction heat normalized to the amount of PafE injected and is corrected for the heat of dilution. The solid line is the nonlinear least-square fitting of the data. n = binding sites; K_d, dissociation constant.

Fig. 4.

Cryo-EM of full-length PafE:20S_OG complexes. (A) Surface-rendered side views of the three complexes that coexisted in solution: Mtb 20S_OG alone without PafE (Left, light blue); 20S_OG CPs capped at both ends by PafE dodecamer rings (Center, gray); and 20S_OG CPs capped at one end by a PafE dodecamer ring (Right, brown). The 3D maps were low-pass–filtered to ∼13 Å. The PafE density is smaller in the singly capped complex, which may reflect increased flexibility. (B) Surface-rendered and semitransparent 3D map of the Mtb 20S_OG in side (Left) and top (Right) views docked with the crystal structure of Mtb 20S proteasome (PDB ID code 3MI0). (C) Superimposition of the 3D maps of Mtb 20S_OG (light blue) and 20S_OG CPs capped at one end by the PafE ring (brown). The substrate entrance in the α-ring of the proteasome was enlarged by ∼30% (6 Å) at the PafE-capped end (Left), whereas the entrance size was unchanged at the uncapped end (Right). (D) Superimposition of the 3D maps of the Mtb 20S_OG (light blue) and Mtb 20S_OG capped at both ends by PafE rings (gray). The substrate entrance gates in the two α-rings of the open-gate proteasome were both enlarged by ∼35% or 7 Å. (Right) A section of the 3D difference map between 20S_OG with and without PafE binding (magenta) superimposed on the 20S_OG map (light blue) in top views. The sectioning plane is marked by the dashed line in the left panel. The difference density peaks from the PafE C termini are located between α-subunits of the proteasome.

Fig. 5.

Shortening PafE C-terminal (CT) linker increases binding affinity to 20S CPs. (A) Zoomed side view of the 3D map of PafE:20S_OG docked with the crystal structures of PafE (magenta) and Mtb 20S (PDB 3MI0, yellow) (Left) or docked with the PA26-proteasome complex structure (PDB ID code 1FNT, purple ribbon) (Right). PA26 is ∼20 Å closer to the proteasome than PafE, indicating an extended linker region between the helical domain and the CT GQYL motif in PafE (magenta dashed lines, Left). (B) PafE has a four-helix bundle fold similar to that of PA26 and PA28 subunits. The PafE CT activation peptide is double the length of that of PA26 and PA28. (C) Schematic diagram of the PafE constructs with different C-terminal domains. (D) SDS/PAGE of gel filtration peaks of 20S-PafE complexes showing the binding of PafE CT peptide truncations to 20S_T1A CPs. (E) Electron micrograph of PafE (Left) or PafE_Δ155–166 (Right) incubated with Mtb 20S_OG CPs. Nearly 90% of the 20S_OG particles were doubly capped by the PafE_Δ155–166 rings. (F) Zoomed side views of 3D maps of 20S CPs (PDB ID code 3MI0, yellow ribbon) docked with WT and PafE truncation mutants (magenta ribbon).

Fig. 6.

PafE mutants with shortened C termini have dramatically increased 20S activation capacity. Peptide degradation assays using PafE truncation mutants. **P < 0.005; ***P < 0.0005; ****P < 0.0001.

Fig. 7.

Cryo-EM of Mtb PafE _Δ155–166:20S_WT complexes. (A) Surface-rendered side views of the three complexes that coexisted in solution: Mtb 20S CP capped at both ends by PafE_Δ155–166 dodecamer rings (Left, light green); Mtb 20S CP alone without PafE_Δ155–166 (Center, gray); and 20S CP capped at one end by a PafE_Δ155–166 dodecamer ring (Right, brown). The 3D maps were low-pass–filtered to ∼12 Å. (B) End-on views of α-rings of proteasome complexes. The substrate entrance in the α-ring of 20S CP was open (∼27 Å in diameter) at the PafE-capped end (Left and Upper Right), whereas the entrance was closed at the uncapped end (Center and Lower Right). (C) Superimposition of the 3D maps of the Mtb 20S CP (gray) and Mtb 20S CP capped at one end by PafE_Δ155–166 rings (brown, Left). (Right) A section of the 3D difference map between 20S with and without PafE_Δ155–166 binding (magenta) superimposed on the 20S CP map (gray) in top views. The dashed line marks the sectioning plane in the Left panel. The difference density peaks from the PafE C termini are located between α-subunits of the proteasome. (D, Left) The PA26 C-terminal motif (PDB ID code 1FNT, magenta spheres), modeled in the predicted Mtb 20S CP activation pocket between two α-subunits (PDB ID code 3MI0, green and gray surfaces, respectively). The difference density, as shown in C, is rendered semitransparent (magenta) for clarity. The amino acid, shown as red spheres, at the bottom of the pocket marked by an arrow is Lys52 in the right α-subunit (gray surface). (Right) PafE-activated peptide degradation activities of Mtb 20S CPs with and without the α-subunit PrcA_K52A mutation.

Fig. 8.

C-terminally truncated PafE variants show increased activity in vivo. (A) Complementation of an Mtb pafE mutant partially restores PafE protein levels. A single copy of WT or truncated pafE was introduced into the chromosome. Total cell lysates were prepared from each strain, separated by 13% (wt/vol) SDS/PAGE, and analyzed by immunoblotting using antibodies raised against PafE. (B) Truncated PafE variants complement the growth defect of a pafE mutant. The indicated strains were diluted in triplicate to OD₅₈₀ = 0.025, and OD₅₈₀ was measured at the indicated time points. (C) A truncated PafE variant fully complements the heat-shock resistance of a pafE mutant. The indicated strains were diluted in triplicate to OD₅₈₀ = 0.08, incubated at 45 °C for 24 h, and inoculated onto Middlebrook 7H11 agar to enumerate surviving bacteria. Statistical analysis was done by nonparametric Student’s t test. For the pafE mutant and complemented strain, an asterisk indicates these strains were significantly more sensitive to heat shock than the parental strain with a P < 0.05, but the strain complemented with WT pafE is significantly improved compared with the mutant (P = 0.0183).