Targeting the cell wall of Mycobacterium tuberculosis: structure and mechanism of L,D-transpeptidase 2

Abstract

With multidrug-resistant cases of tuberculosis increasing globally, better antibiotic drugs and novel drug targets are becoming an urgent need. Traditional β-lactam antibiotics that inhibit D,D-transpeptidases are not effective against mycobacteria, in part because mycobacteria rely mostly on L,D-transpeptidases for biosynthesis and maintenance of their peptidoglycan layer. This reliance plays a major role in drug resistance and persistence of Mycobacterium tuberculosis (Mtb) infections. The crystal structure at 1.7 Å resolution of the Mtb L,D-transpeptidase Ldt(Mt2) containing a bound peptidoglycan fragment, reported here, provides information about catalytic site organization as well as substrate recognition by the enzyme. Based on our structural, kinetic, and calorimetric data, we propose a catalytic mechanism for Ldt(Mt2) in which both acyl-acceptor and acyl-donor substrates reach the catalytic site from the same, rather than different, entrances. Together, this information provides vital insights to facilitate development of drugs targeting this validated yet unexploited enzyme.

Figure 1

Peptidoglycan linkages and structure of Ldt_Mt2. (A) 4→3 and 3→3 linkages. The C-terminal residues from the γ-D-Glu of donor and acceptor peptidoglycan stems are depicted in different colors, γ-D-Glu (red), m-A₂pm (blue), and D-Ala (cyan). Residue numbers of the acceptor stem are primed. (B) Cartoon representation of the overall structure of ex-Ldt_Mt2 with elements of secondary structure labeled. Two pink arrows mark the beginning and end of the CTSD. The peptidoglycan fragment bound to the active site is also shown in a stick representation. (C and D) Views of the solvent accessible surface of ex-Ldt_Mt2 colored by electrostatic surface-potential (from blue positive to red negative). (C) side view (same orientation as part (B). The stick representation of peptidoglycan-fragment bound to the active site is shown. Oxygen atoms are colored in red, nitrogen atoms in blue, sulfur atoms in yellow and carbon atoms in green. (D) top view of the outer cavity (rotated 90° around a horizontal axis from B). The surface was rendered transparent and the peptidoglycan fragment removed to show residues and secondary structure elements lining the outer cavity.

Figure 2

Sequence alignment of Ldt_Mt2, the L,D-transpeptidase of E. faecium (1ZAT) and other Mtb Ldt_Mt2 homologues_. Top: domain distribution of Ldt_Mt2. The ex-Ldt_Mt2 protein construct used in this work is indicated by a dashed red box. Bottom: sequence alignment among amino acids 138 to 408 of Ldt_Mt2, Ldt_MT1, Ldt_fm and other Ldt_Mt2 homologues in Mtb. The observed secondary structure is indicated above the corresponding residues. Identical residues are white with red background, regions scoring higher than 70 % using a Risler’s similarity scoring-matrix (Risler et al., 1988) are boxed with red letters. Residues involved in binding and catalysis are labeled: the conserved motif is indicated with green marks and the rest with pink marks. Stars label main catalytic residues and ovals label substrate recognition residues. The figure was made using the web server ESPript (http://espript.ibcp.fr/ESPript/ESPript/).

Figure 3

Comparative Modeling of Mtb homologues. (A) Overlay of Cα-trace of the modeled structures of the Mtb homologues with the crystal structure of ex-Ldt_Mt2 (green). (B) Overlay of the modeled catalytic sites. Residues participating in the interactions with the substrate are displayed; carbons atoms are colored with the same color as the corresponding Cα-trace: Green, Ldt_Mt2; yellow, MT1477; pink, MT0501; magenta, Ldt_Mt1; and cyan, MT0202. Non-carbon atoms are colored as Fig. 1.

Figure 4

Electron densities at the catalytic site of Ldt_Mt2. (A) Final refinement 2DF_o - mF_c map of the co-crystallized peptidoglycan fragment contoured at 0.5 σ (see below) around the γ-D-Glu-m-A₂pm fragment at the active site of ex-Ldt_Mt2. The fitted fragment, binding residues and the catalytic cysteine are shown. Hydrogen bond interactions with conserved residues are shown with pink dashed lines. As the two binding sites in the non-crystallographic dimer occur side-by-side, overlapping density for two equivalent peptidoglycan fragments was observed. The density was interpreted as two equivalent ligands related by the non-crystallographic two fold and refined as alternative orientations with occupancy of 0.5. (See also Fig. S2 for a stereo view of an omit map). (B-C) Modifications of Cys354 observed in native forms. (B) β-mercapto-ethanol covalently bound to Cys354 (2.0 Å bond distance). The rms deviation between this structure (native form) and the refined (PIP derivative) is 0.176 Å for 520 Cα-atoms of the asymmetric unit. Final refinement 2DF_o – mF_c electron density map contoured at 1 sigma around Cys354. (C) Oxidized Cys354 (suphenic acid form). Atoms are colored as Fig. 1.

Figure 5

Isothermal titration calorimetry experiments. The chemical structures of (A) Imipenem ((5R,6S)-6-[(1R)-1-hydroxymethyl]-3-({2-[iminomethyl)amino]ethyl}thio)-7-oxo-1 azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid), and (B) meropenem (3-[5-(dimethylcarbamoyl) pyrrolidin-2-yl] sulfanyl-6-(1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0]-hept-2-ene-2-carboxylic acid) are shown. Titrations of (C) imipenem and (D) meropenem binding to Ldt_Mt2 are shown in the upper portion of the panels. The lower portion of the panel displays plots of the total heat released as a function of the molar ratio between each carbapenem and Ldt_Mt2. The solid line represents the non-linear least square fit of the data using a single-site binding model. Thermodynamic parameters of binding to ex-Ldt_Mt2 --K_D, ΔH and ΔS-- are tabulated at the bottom of the figure.

Figure 6

C354A mutant. (A) ITC of Imipenem binding to the C354A mutant. Upper panel: Titration of the C354 mutant with imipenem. Lower panel: plot of the total heat released as a function of total ligand concentration for the titration shown in the upper panel. No heat of binding is detected, only heat of dilution. (B) Refined electron density of the C354A mutant around the mutated residue. Final refinement 2DF_o – mF_c map contoured at 1 σ around Ala354 of the mutant. Carbons atoms are colored in cyan, the rest as Fig. 1.

Figure 7

Enzymatic activity of Ldt_Mt2. (A) and (B) Irreversible inhibition by iminopenem. (A) three selected concentrations of enzyme (10, 35 and 60 μM) reacting with 100 μM of imipenem. (B) Plot of the concentration of imipenem reacted versus the concentration of enzyme used (10, 20, 35, 40, 50 and 60 μM). (C) Initial-rate experiments of the ex-Ldt_Mt2 β-lactamase activity using nitrocefin as substrate (structural formula show in the Figure). Solid lines are the non-linear fit of the initial rate to the Michaelis-Menten equation. (Note that pH 7 and 8.5, and 6.5 and 9 overlap.) (D) Plot of the log of k_cat for nitrocefin as a function of pH. See also Table S2. (E-F) Progress of the reaction of nitrocefin (100 μM) and Ldt_Mt2 (500 nM). (E) Initial phase of the reaction (controlled by binding and acylation rates). (F) Later phase of the reaction (controlled by de-acylation rate). The amount of product (open ring) is monitored by its absorption at 486 nM.

Figure 8

Structural comparison among L,D transpeptidases from B. subtilus, E. faecium and M. tuberculosis. (A) Side by side ribbon diagrams of the crystal structures of the L,D-transpeptidases: ykuD (PDB id. 1Y7M) in pink (rms deviation of 0.97 Å between 104 Cα-atoms aligned with ex-Ldt_Mt2), Ldt_fm (PDB id. 1ZAT) in cyan (1.0 Å rms deviation between 116 Cα-atoms aligned) and Ldt_Mt2 in green. In the E faecium and Mtb case, the regions that apparently fix the domains orientation are colored in dark green. The catalytic domains of the three enzymes were used to align the structures. (B) Comparison of the catalytic domains. The catalytic cysteine residue is depicted in a CPK representation with carbon atoms purple and sulphur atom yellow. (C) Catalytic site residues of the three enzymes. View them from the outer cavity; the catalytic site residues are shown as side-chains or main-chains according to their expected function. The catalytic site triad is labeled with pink letters. The γ-glutamyl-meso-diaminopymelic acid (γ-D-Glu-m-A₂pm) is shown only for ldt_Mt2 in a stick representation with magenta colored carbon atoms. Non-carbon atoms are colored as Fig. 1. H-bond interactions of the peptidoglycan fragment with residues of the catalytic site are shown with dashed lines.

Figure 9

Proposed mechanism of the L,D-transpeptidation reaction and structural model of the acyl-acceptor and donor-peptidoglycan stems bound to the outer cavity of ex-Ldt_Mt2. (A) Steps 1 – 6 of the mechanism of (3,3) L,D-transpeptidation by Ldt_Mt2. (B) The figure represents step 4 of the mechanism in part A, after the cleaved D-Ala⁴ has left and a thioester intermediate is formed between acyl-donor (carbon atoms colored pink) and the enzyme (cyan). Non-carbon atoms are colored as Fig. 1. Only the last two residues of the donor stem and the m-A₂pm (light gray) of the acceptor stem are shown. The peptidoglycan stem of the acyl-acceptor binds to the site with its A₂pm3′ side chain (D chiral-center) near the thioester bond (marked with an *). (B) Solvent accessible surface at the outer cavity. The surface is colored by electrostatic surface-potential (from blue positive to red negative). The two substrates of the bi-substrate reaction are shown.