Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation

Abstract

Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an α-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

Figure 1

Mpa1-234 hexamer has three 75 Å long coiled-coils needed for Pup recognition. (a) Crystal structure of Mpa1-234 revealed three long coiled-coils formed by six helices that sit atop the hexameric double OB-fold domain. Mpa1-46 was disordered in the crystal structure, and is thus not shown. (b) Two orthogonal views of the Mpa coiled-coil in cartoon representation. The coiled-coil was stabilized via a leucine zipper-like mechanism. Ala80 replaces the expected leucine at the 4^th heptad repeat position. Presence of two adjacent leucines at positions 84 and 85 forced Leu85 to face the solvent. (c) Cartoon view of the co-crystal structure of the Pup21-64 in complex with the Mpa46-96 coiled-coil. Pup folds into an α-helix upon binding to the C-terminal half of the coiled-coil formed by Mpa-Ha and Mpa-Hb (see text for details). Arrows indicate helix direction. (d) Surface view of Pup21-64 on the Mpa46-96 coiled-coil (left). “Pulled apart” view of Pup21-64 and the Mpa46-96 coils (right). Hydrophobic residues in Pup that interact with Mpa Ha are colored in orange, and with Mpa Hb in yellow. The conserved Pup Ile43 (red) was centrally located and interacted with residues in both the Ha and Hb helices of Mpa. (e) The electron density map revealed that the conserved Asn50 of Pup and Asn70 of Mpa assume dual conformations. The conformations of Mpa Asn70 and Pup Asn50 enable H-bond formation within the Mpa coiled-coil and between Pup and Mpa. (f) Several hydrogen bonds further stabilize the chiefly hydrophobic interaction between Mpa (blue) and Pup (cyan). The σ_A-weighted 2Fo-Fc density map is contoured at 1σlevelaroundtheconservedresidues.

Figure 2

Full-length Pup in the context of hexameric Mpa1-234. (a) Overall structure of the Pup:Mpa1-234 complex showing three Pups (red) apparently bound to all three Mpa coiled-coils. The red arrows point to the cross loops of the OB folds, which partially block the outside surfaces of the Mpa coiled-coils and prevent Pup binding. Pup formed an α-helix upon binding to the hexameric Mpa1-234, and interacted with the Mpa coiled-coil in an anti-parallel fashion, as observed in the Pup21-64:Mpa46-96 structure. Mpa1-51, Pup1-20, and Pup52-64 were disordered in the Pup:Mpa1-234 complex structure. (b) Alignment at the Mpa coiled-coil region of Pup:Mpa1-234 (magenta) with that of Pup21-64:Mpa46-96 (green) revealed that the presence of Pup1-20 in the hexameric structure resulted in the N-terminus of the Pup helix to point away from the Mpa coiled-coil (red arrows). This movement can be approximated by the Pup helix tilt at about 4° around its C-terminal end (dashed lines). (c) High-resolution view of Pup with the Mpa1-234 coiled-coil. Pup (cyan) is shown in cartoon view and hexameric Mpa is shown in surface charge view. The positively charged middle region of the Mpa coiled-coil was neutralized by the negatively charged C-terminal half of the Pup helix (the black boxed region of the middle panel and in the left panel). Conversely, two arginines (28 and 29) in Pup neutralized the two negatively charged pockets at the root of the Mpa coiled-coil (white boxed region in the middle panel, enlarged on the right).

Figure 3

Essentiality of the Pup helical region for proteasomal degradation supports a binding-induced folding recognition mechanism by Mpa. (a) Site-directed mutations in Pup resulted in abrogated degradation of a Pup-linear fusion by the Msm proteasome. The binding-induced helical region is in red. Mutated residues are indicated. (b) Equivalent cell numbers were analyzed from stationary phase cultures of wild type (WT) or proteasome-deleted (ΔprcBA, ΔBA) M. smegmatis synthesizing WT or mutated Pup-Zur-His₆. Detection of the linear fusion proteins was done with anti-His₅. DlaT (dihydrolipoamide acyltransferase) was the loading control. (c) Model for the targeting of pupylated proteins for degradation by Mpa and the mycobacterial proteasome. The Pup:Mpa1-234 complex structure (red and cyan) was placed over the homologous PAN AAA+ domain structure (PDB ID 3H4M, magenta), which was further overlaid on the Mtb proteasome core structure (PDB ID 2FHH, gray). Only a vertical central slice of the complex structure is shown for clarity. Pup is in red, and a model substrate (GFP) in green. See main text for details.