Structural Analysis of the Bacterial Proteasome Activator Bpa in Complex with the 20S Proteasome

Abstract

Mycobacterium tuberculosis harbors proteasomes that recruit substrates for degradation through an ubiquitin-like modification pathway. Recently, a non-ATPase activator termed Bpa (bacterial proteasome activator) was shown to support an alternate proteasomal degradation pathway. Here, we present the cryo-electron microscopy (cryo-EM) structure of Bpa in complex with the 20S core particle (CP). For docking into the cryo-EM density, we solved the X-ray structure of Bpa, showing that it forms tight four-helix bundles arranged into a 12-membered ring with a 40 Å wide central pore and the C-terminal helix of each protomer protruding from the ring. The Bpa model was fitted into the cryo-EM map of the Bpa-CP complex, revealing its architecture and striking symmetry mismatch. The Bpa-CP interface was resolved to 3.5 Å, showing the interactions between the C-terminal GQYL motif of Bpa and the proteasome α-rings. This docking mode is related to the one observed for eukaryotic activators with features specific to the bacterial complex.

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

“…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.…”

“…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).…”

“…Furthermore, the sitespecific cross-linker para-benzoyl-Lphenylalanine was used in place of threonine-170 in PafE, a residue immediately preceding the GQYL motif. Conceivably, 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). While these previous studies were informative, we felt that more details on the mechanism of proteasome gate opening by PafE remained to be elucidated.…”

AAA+ Machines of Protein Destruction in Mycobacteria

“…Following this step, the substrate is translocated through the central pore of the unfoldase (in an ATP-dependent manner), into the proteolytic chamber of the associated peptidase where the substrate is cleaved into small peptide fragments. Interestingly, in some cases these peptidases are also activated for the energy-independent turnover of specific protein substrates, through the interaction with non-AAA+ components (Bai et al, 2016; Bolten et al, 2016). These nucleotide-independent components facilitate substrate entry into the proteolytic chamber by opening the gate into the peptidases, as such we refer to them as gated dock-and-activate (GDA) proteases.…”

AAA-ATPases in Protein Degradation

“…The alpha ring gate of mycobacterial proteasomes can also be widened by Bpa, a just recently identified non-ATPase ring, suggesting that the AAA-ATPase activity is not required for alpha ring gating (Bolten et al, 2016). …”