Inactivation of the Mycobacterium tuberculosis antigen 85 complex by covalent, allosteric inhibitors

Abstract

The rise of multidrug-resistant and totally drug-resistant tuberculosis and the association with an increasing number of HIV-positive patients developing tuberculosis emphasize the necessity to find new antitubercular targets and drugs. The antigen 85 (Ag85) complex from Mycobacterium tuberculosis plays important roles in the biosynthesis of major components of the mycobacterial cell envelope. For this reason, Ag85 has emerged as an attractive drug target. Recently, ebselen was identified as an effective inhibitor of the Ag85 complex through covalent modification of a cysteine residue proximal to the Ag85 active site and is therefore a covalent, allosteric inhibitor. To expand the understanding of this process, we have solved the x-ray crystal structures of Ag85C covalently modified with ebselen and other thiol-reactive compounds, p-chloromercuribenzoic acid and iodoacetamide, as well as the structure of a cysteine to glycine mutant. All four structures confirm that chemical modification or mutation at this particular cysteine residue leads to the disruption of the active site hydrogen-bonded network essential for Ag85 catalysis. We also describe x-ray crystal structures of Ag85C single mutants within the catalytic triad and show that a mutation of any one of these three residues promotes the same conformational change observed in the cysteine-modified forms. These results provide evidence for active site dynamics that may afford new strategies for the development of selective and potent Ag85 inhibitors.

Keywords: Antigen 85 Complex; Carbohydrate; Drug Discovery; Enzyme Inactivation; Mycobacterium tuberculosis; Protein Structure.

FIGURE 1.

Native Ag85C structure (PDB 1DQZ). A, overall structure of Ag85C. The α helices are colored in green, and the β strands are colored in wheat, respectively. B, active site of the native Ag85C structure. The catalytic triad residues (Ser-124, Glu-228, and His-260), as well as Cys-209 and Thr-231/Leu-232, are represented in sticks. Cys-209 is positioned on strand β7 and interacts through van der Waals interactions with the peptide bond linking Thr-231 to Leu-232 located on helix α9; the interactions maintain the helix α9 in a bent conformation. Helix α9 also harbors Glu-228, one of the residues of the catalytic triad participating in the Ag85 mechanism.

FIGURE 2.

A, superposition of the native Ag85C (gray), Ag85C-EBS (orange), and Ag85C-IAA (green) structures. The difference density of a F_o − F_c omit map is shown contoured at 3σ (green); the side chain of the YCM residue was omitted during map calculation. B, superimposition of the native Ag85C (gray), Ag85C-EBS (orange), and Ag85C-Hg (blue) structures. The difference density of a F_o − F_c omit map is shown contoured at 3σ (blue); the side chain of the CHg residue was omitted during map calculation. CHg represents the Cys-209 residue modified with mercuribenzoic acid.

FIGURE 3.

Inhibition of Ag85C by p-chloromercuribenzoic acid. The enzymatic activity of the different samples was tested using a fluorescence-based assay. The activity is normalized to the unmodified Ag85C enzyme, and the error bars are calculated from triplicate reactions. A, inhibition of Ag85C by p-chloromercuribenzoic acid after a 2-h incubation. Bar 1 corresponds to the unmodified Ag85C, whereas bar 2 relates to the Ag85C enzyme modified with p-chloromercuribenzoic acid. B, inhibition of Ag85C by p-chloromercuribenzoic acid after an overnight incubation. Bar 1 corresponds to the unmodified Ag85C, whereas bar 2 relates to the Ag85C enzyme modified with p-chloromercuribenzoic acid. Error bars correspond to S.D. from triplicate reactions.

FIGURE 4.

Ag85C-ebselen structure. A, proposed reaction mechanism of cysteine modification with ebselen. B, superposition of the new Ag85C-ebselen (dark orange) and Ag85C-EBS (orange) structures. The electron density of an F_o − F_c omit map is shown contoured at 3σ (gray); the side chain of the Ceb (Cys-209 modified by ebselen) residue was omitted during map calculation. Electron density was only observed for the exocyclic selenium atom, the first aromatic ring, and the amide linker. C, Ag85C-ebselen structure (bright orange). The electron density of an anomalous map is shown contoured at 4σ (blue).

FIGURE 5.

Superposition of Ag85C (gray), Ag85C-EBS (orange), Ag85C-C209S (yellow), and Ag85C-C209G (cyan) structures. The His-260 side chain interacts with the serine nucleophile Ser-124 in the native structure, whereas it interacts with a different serine residue (Ser-148) in the Ag85C-C209G structure, explaining the lack of activity displayed by the mutant. The mutation promotes the relaxation of helix α9, similar to the structural change observed in the Ag85C-C209S or Cys-209 covalently modified Ag85C structures.

FIGURE 6.

Effect of mutations among the catalytic triad residues. The enzymatic activity of the mutants was tested using a fluorometric assay. The activity was normalized to the wild-type enzyme Ag85C, and the error bars were calculated from triplicate reactions. A, the mutant Ag85C-H260Q exhibits less than 10% of activity compared with the wild-type Ag85C. B, Ag85C-E228Q retains 17% of wild-type activity. Error bars correspond to S.D. from triplicate reactions.

FIGURE 7.

Superposition of Ag85C-S124A (orange), Ag85C-E228Q (green), and Ag85C-H260Q (blue) structures. The three mutants exhibit a relaxation of helix α9, accounting for the loss of enzymatic activity.