The mycobacterial Mpa–proteasome unfolds and degrades pupylated substrates by engaging Pup's N‐terminus

Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin‐like protein (Pup) that is recognized by the N‐terminal coiled‐coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substrates and establish its mechanistic features with respect to substrate recruitment, unfolding and degradation. We show that the Mpa–proteasome complex unfolds and degrades Pup‐tagged proteins and that this activity requires physical interaction of the ATPase with the proteasome. Furthermore, we establish the N‐terminal region of Pup as the structural element required for engagement of pupylated substrates into the Mpa pore. In this process, Mpa pulls on Pup to initiate unfolding of substrate proteins and to drag them toward the proteasome chamber. Unlike the eukaryotic ubiquitin, Pup is not recycled but degraded with the substrate. This assigns a dual function to Pup as both the Mpa recognition element as well as the threading determinant.     

The pupylation pathway and its role in mycobacteria

Pupylation is a post-translational protein modification occurring in actinobacteria through which the small, intrinsically disordered protein Pup (prokaryotic ubiquitin-like protein) is conjugated to lysine residues of proteins, marking them for proteasomal degradation. Although functionally related to ubiquitination, pupylation is carried out by different enzymes that are evolutionarily linked to bacterial carboxylate-amine ligases. Here, we compare the mechanism of Pup-conjugation to target proteins with ubiquitination, describe the evolutionary emergence of pupylation and discuss the importance of this pathway for survival of Mycobacterium tuberculosis in the host.     

Deletion of dop in Mycobacterium smegmatis abolishes pupylation of protein substrates in vivo

Proteasome‐bearing bacteria make use of a ubiquitin‐like modification pathway to target proteins for proteasomal turnover. In a process termed pupylation, proteasomal substrates are covalently modified with the small protein Pup that serves as a degradation signal. Pup is attached to substrate proteins by action of PafA. Prior to its attachment, Pup needs to undergo deamidation at its C‐terminal residue, converting glutamine to glutamate. This step is catalysed in vitro by Dop. In order to characterize Dop activity in vivo, we generated a dop deletion mutant in Mycobacterium smegmatis. In the Δdop strain, pupylation is severely impaired and the steady‐state levels of two known proteasomal substrates are drastically increased. Pupylation can be re‐established by complementing the mutant with either DopWt or a Pup variant carrying a glutamate at its ultimate C‐terminal position (PupGGE). Our data show that Pup is deamidated by Dop in vivo and that likely Dop alone is responsible for this activity. Furthermore, we demonstrate that a putative N‐terminal ATP‐binding motif is crucial for catalysis, as a single point mutation (E10A) in this motif abolishes Dop activity both in vivo and in vitro.     

Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB

Cell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins.     

Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated pup intermediate

In Mycobacterium tuberculosis, the enzyme PafA is responsible for the activation and conjugation of the proteasome-targeting molecule Pup to protein substrates. As the proteasomal pathway has been shown to be vital to the persistence of M. tuberculosis, understanding the reaction mechanism of PafA is critical to the design of antituberculous agents. In this study, we have developed novel techniques to study the activity of PafA and have characterized fundamental features of the reaction mechanism. We show that PafA catalyzes a two-step reaction mechanism proceeding through a γ-glutamyl phosphate-mixed anhydride intermediate that is formed on the C-terminal glutamate of Pup before transfer of Pup to the substrate acceptor lysine. SDS-PAGE analysis of formation of the phosphorylated intermediate revealed that the rate of Pup activation matched the maximal steady-state rate of product formation in the overall reaction and suggested that Pup activation was rate-limiting when all substrates were present at saturating concentrations. Following activation, both ADP and the phosphorylated intermediate remained associated with the enzyme awaiting nucleophilic attack by a lysine residue of the target protein. The PafA reaction mechanism appeared to be noticeably biased toward the stable activation of Pup in the absence of additional substrate and required very low concentrations of ATP and Pup relative to other carboxylate-amine/ammonia ligase family members. The bona fide nucleophilic substrate PanB showed a 3 orders of magnitude stronger affinity than free lysine, promoting Pup conjugation to occur close to the rate limit of activation with physiologically relevant concentrations of substrate.     

Assembly pathway of an AAA+ protein: tracking ClpA and ClpAP complex formation in real time

The ClpAP chaperone-protease complex is active as a cylindrically shaped oligomeric complex built of the proteolytic ClpP double ring as the core of the complex and two ClpA hexamers associating with the ends of the core cylinder. The ClpA chaperone belongs to the larger family of AAA+ ATPases and is responsible for preparing protein substrates for degradation by ClpP. Here, we study in real time using fluorescence and light scattering stopped-flow methods the complete assembly pathway of this bacterial chaperone-protease complex consisting of ATP-induced ClpA hexamer formation and the subsequent association of ClpA hexamers with the ClpP core cylinder. We provide evidence that ClpA assembles into hexamers via a tetrameric intermediate and that hexamerization coincides with the appearance of ATPase activity. While ATP-induced oligomerization of ClpA is a prerequisite for binding of ClpA to ClpP, the kinetics of ClpA hexamer formation are not influenced by the presence of ClpP. Models for ClpA hexamerization and ClpA-ClpP association are presented along with rate parameters obtained from numerical fitting procedures. The hexamerization kinetics show that the tetrameric intermediate transiently accumulates, forming rapidly at early time points and then decaying at a slower rate to generate the hexamer. The association of assembled ClpA hexamers with the ClpP core cylinder displays cooperativity, supporting the coexistence of interchanging ClpP conformations with different affinities for ClpA.     

The flexible attachment of the N-domains to the ClpA ring body allows their use on demand

ClpA is an Hsp100 chaperone that uses the chemical energy of ATP to remodel various protein substrates to prepare them for degradation. It comprises two AAA+ modules and the N-domain, which is attached N-terminally to the first AAA+ module through a linker. On the basis of cryo-electron microscopic and X-ray crystallographic data it has been suggested that the linker confers mobility to the N-domain. In order to define the role of the N-domain in ClpAP-dependent substrate degradation we have generated a ΔN variant at the protein level by introducing a protease cleavage site. The ClpA molecule generated in this way lacks the N-domain and the associated linker but is impaired only slightly in the processing of substrates that are degraded independently of ClpS. In fact, it shows increased catalytic efficiency in the degradation of ssrA-tagged GFP compared to ClpAwt. The role of the linker attaching the N-domain to the bulk of the molecule was probed by characterizing variants with different lengths of the linker. The degradation efficiency of a ClpS-dependent N-end rule substrate, FRliGFP, is reduced for linkers that are shorter or longer than natural linkers but remains the same for the variant where the linker is replaced by an engineered sequence of equivalent length. These results suggest that the flexible attachment of the N-domains to ClpA allows their recruitment to the pore on demand for certain substrates, while allowing them to move out of the way for substrates binding directly to the pore.     

Characterization of a new AAA+ protein from archaea

We investigated a new archaeal member of the AAA+ protein family (ATPases associated with various cellular activities) which is found in all methanogenic archaea and the sulphate-reducer Archaeoglobus fulgidus. These proteins cluster to COG1223 predicted to form a subgroup of the AAA+ ATPases. The gene from A. fulgidus codes for a protein of 40 kDa monomeric molecular weight, which we overexpressed in Escherichia coli and purified to homogeneity. The protein forms ring-shaped complexes with a diameter of 125A as determined by electron microscopy. Using sedimentation equilibrium analysis we demonstrate that it assembles into hexamers over a wide concentration range both in presence and absence of ATP. As suggested by homology to other members of the AAA+ family, the complex binds and hydrolyzes ATP. Michaelis-Menten analysis revealed a k(cat) of 118 min(-1) and a K(M) of 1.4 mM at 78 degrees C. This hyperthermophilic archaeal ATPase is stable to 86 degrees C and the ATPase activity is maximal at this temperature. The protein is most homologous to the AAA-domain of FtsH from bacteria, while the N-terminal domain shows predicted structural homology to members of the CDC48 family of AAA proteins. Possible roles of this new AAA+ protein are discussed.