Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-deconjugating enzyme is an unusual aspartate amidase

Abstract

Deamidase of Pup (Dop), the prokaryotic ubiquitin-like protein (Pup)-deconjugating enzyme, is critical for the full virulence of Mycobacterium tuberculosis and is unique to bacteria, providing an ideal target for the development of selective chemotherapies. We used a combination of genetics and chemical biology to characterize the mechanism of depupylation. We identified an aspartate as a potential nucleophile in the active site of Dop, suggesting a novel protease activity to target for inhibitor development.

FIGURE 1.

A, pupylation pathway in mycobacteria. B, general strategy for the formation of the HA-Pup-DON trap. HA-tagged Pup was cloned in-frame with an intein domain fused to a chitin-binding domain (CBD), and the HA-PupΔ_Gln was overproduced and bound to chitin resin. The bound protein was incubated with MeSNa, resulting in the elution of HA-Pup-MeSNa. Addition of DON to the reaction mixture displaced MeSNa and generated HA-Pup-DON. C, Dop immunoblot (IB) of the reaction with HA-Pup-DON and lysates of M. tuberculosis WT, dop, and dop-complemented strains. All strains contain the integrative plasmid pMV306 (empty vector) or pMV306 with WT dop or dopE10A at the attB site of the chromosome. In C and F, the asterisk indicates a species only seen in the dop:ΦMycoMarT7 mutant and is predicted to be truncated Dop. Samples were separated on a 9% SDS-PAGE gel. D, HA-Pup-DON reaction with purified Dop. Slowed migration of Mycobacterium smegmatis Dop (left) and Mycobacterium tuberculosis Dop (right) only occurred in the presence of HA-Pup-DON and ATP. Samples were separated on a 12% SDS-PAGE gel. Arrowhead indicates HA-Pup-DON∼Dop. The faster migrating species is specific to the Pup-DON trap as it was recognized alone by antibodies to the HA epitope (center lane). E, high resolution tandem mass spectrometry analysis of the HA-immunoprecipitated HA-Pup-DON∼Dop species indicating a modification of mass 257.1 Da on Asp-95 of Dop. F, Dop immunoblot of the reaction with HA-Pup-DON trap and lysates of M. tuberculosis WT, dop, and dop-complemented strains. Samples were separated on a 10% SDS-PAGE gel. G, proposed mechanism of HA-Pup-DON and Dop conjugation reaction. HA-Pup-DON is protonated in the active site of Dop, followed by a nucleophilic attack to displace nitrogen, and this results in the covalent HA-Pup-DON∼Dop adduct.

FIGURE 2.

A, proposed model of the Dop active site, based on the crystal structure of E. coli YbdK (PDB code 1R8G) (24). Dop is colored green, and select side chains are shown in stick representation. Two Mg²⁺ ions (from PDB code 1VA6 (γ-glutamyl-cysteine synthetase)) are shown as magenta spheres. Oxygen atoms are colored red, and nitrogen atoms are colored blue. ADP is shown in stick representation with carbon atoms colored yellow and phosphorus atoms colored gray. The modeled Pup∼substrate (Lys) is colored orange (carbon atoms) with the terminal glutamate and conjugated lysine shown in stick representation. B, mutational analysis of potential Dop nucleophiles. Total cell lysates from equivalent cell numbers of a M. tuberculosis dop mutant strain containing integrated empty vector or plasmids encoding either wild type (WT) or mutated dop alleles were separated on a 10% SDS-PAGE gel and analyzed for pupylation by immunoblotting (IB) with monoclonal antibodies to M. tuberculosis Pup. The membrane was stripped and incubated with antibodies to Dop (below).

FIGURE 3.

¹⁸O-Incorporation by mass spectrometry. The Dop-catalyzed Pup∼Ino1 depupylation reaction was monitored in the presence of 50% ¹⁸O-water, showing a shift in the isotopic distribution of the C-terminal Pup_Glu peptide corresponding to ¹⁸O-incorporation. B, ¹⁸O-incorporation not identified on Dop. C, Dop reaction with hydroxylamine. Chitin-binding domain is stained with 16% SDS-PAGE of the Pup∼Ino1 depupylation reaction: (left) in the absence of hydroxylamine; (center) in the presence of hydroxylamine; and (right) in the presence of hydroxylamine without ATP. Middle panel, enhanced image of the 10–12-kDa region of the gel above. Bottom, Pup IB of the two species formed upon addition of hydroxylamine is shown. D, Pup_Glu after incubation with hydroxylamine. Chitin-binding domain is stained with 16% SDS-PAGE of Pup reaction with Dop: (left) in the absence of hydroxylamine; (center) in the presence of hydroxylamine; and (right) in the presence of hydroxylamine without Dop. Note that this panel was enhanced to view all possible Pup species. E, Pup-hydroxymate (Pup-NHOH). LC chromatogram of the Pup 1 and Pup 2 species (labeled in C) is shown. Inset, presumed structure of Pup-NHOH. F, proposed mechanism of the Dop-catalyzed deconjugation reactions. The aspartate nucleophile attacks the Pup∼substrate amide, forming an anhydride intermediate. Base-catalyzed deprotonation of a water molecule activates it to attack at the Pup side of the anhydride to form the products.