The pup-proteasome system of Mycobacterium tuberculosis

Abstract

Proteasomes are ATP-dependent protein degradation machines present in all archaea and eukaryotes, and found in several bacterial species of the order Actinomycetales. Mycobacterium tuberculosis (Mtb), an Actinomycete pathogenic to humans, requires proteasome function to cause disease. In this chapter, we describe what is currently understood about the biochemistry of the Mtb proteasome and its role in virulence. The characterization of the Mtb proteasome has led to the discovery that proteins can be targeted for degradation by a small protein modifier in bacteria as they are in eukaryotes. Furthermore, the understanding of proteasome function in Mtb has helped reveal new insight into how the host battles infections.

Fig. 10.1. Structure of the Mtb 20S core particle (CP)

(a) Side view of the Mtb 20S CP structure rendered in cartoon view (PDB ID 2FHG). The two α-rings at the top and the bottom are displayed in green, and the two catalytic middle β-rings in blue. (b) The same side view as in (a), but the structure is rendered in surface display. Only a central slab of 30 Å is shown such that the two anterior chambers and one central chamber of the CP are visible. The red circles mark the proteolytic sites

Fig. 10.2. Top view of the Mtb 20S CP

(a) and (b) Closed gate conformation (PDB ID 3MKA). The gate formed by the α-subunits is ordered and closed with a mechanism different to the eukaryotic CP. The seven subunits are chemically identical but adopt a total of three different conformations at their N-termini as indicated by the different colors in (a). A phenylalanine side chain from each of the green octapeptides contributes to the gate closure. (c) Deletion of the N-terminal residues 2–9 results in the open gate conformation (PDB ID 3MFE)

Fig. 10.3. Comparison of Mpa with PAN

(a) Linear map of the domain structure of Mpa. CC = coiled coil; OB = oligosaccharide/oligonucleotide binding domain. (b) The structures of Mtb Mpa1-234 (PDB ID 3M9B), Archaeoglobus fulgidus PAN-ΔCC_CC_GCN4 hybrid (PDB ID 2WG5), and M. jannaschii PAN (PDB ID 3H43) are aligned and displayed individually (Figure adapted from [54])

Fig. 10.4. Eukaryotic ubiquitin-proteasome system

Ubiquitin (Ub) is encoded as part of a larger polypeptide. Proteases expose a C-terminal Gly-Gly motif that is activated by adenylation with an E1 enzyme. The E1 enzyme transfers Ub to an E2 enzyme, where a thioester bond is formed. The E2 then transfers Ub to any number of E3 ligases. The E3 ligase family can be sub-divided into HECT and RING domain ligases: RING ligases interact with both the E2 and substrate, and facilitate the direct transfer of Ub from the E2 to the substrate. In contrast, HECT ligases form a thioester bond with Ub prior to transfer to a substrate Lys. E3 ligases dictate the type of Ub linkages that are formed. Ubiquitylated protein with Lys48 (K48) linked Ub chains are targeted for degradation by the 26S proteasome. Other types of Ub linkages (mono- and poly-K63 and others) generally do not result in degradation but serve other functions

Fig. 10.5. Overview of the Pup-proteasome system in mycobacteria

Pup is deamidated at the C-terminal Gln by Dop. PafA phosphorylates the α-carboxylate of the C-terminal Glu of Pup, priming it for attack by the ε-amino group of a substrate Lys. Pup can be removed by Dop prior to degradation to potentially rescue a substrate from destruction or possibly facilitate its degradation

Fig. 10.6. Binding of Pup to Mpa induces a helical structure at the C-terminus of Pup

(a) Top view of the crystal structure of the hexameric Mpa1-234 complex with Pup (PDB ID 3M9D). The binding-induced Pup helix is shown in the cartoon view in red, and the Mpa coiled-coil and OB domains are shown in surface view in green. (b) Model for the targeting of pupylated proteins for degradation by Mpa and CP. The Pup:Mpa1-234 complex structure (red and green) was placed over the homologous PAN AAA+domain structure (PDB ID 3H4M, magenta), which was overlaid on the Mtb CP (PDB ID 2FHH, yellow). A vertical central slice of the complex structure is shown for clarity. Pup is in red, and a model substrate in green