Structural basis for the inhibition of Mycobacterium tuberculosis L,D-transpeptidase by meropenem, a drug effective against extensively drug-resistant strains

Abstract

Difficulty in the treatment of tuberculosis and growing drug resistance in Mycobacterium tuberculosis (Mtb) are a global health issue. Carbapenems inactivate L,D-transpeptidases; meropenem, when administered with clavulanate, showed in vivo activity against extensively drug-resistant Mtb strains. LdtMt2 (Rv2518c), one of two functional L,D-transpeptidases in Mtb, is predominantly expressed over LdtMt1 (Rv0116c). Here, the crystal structure of N-terminally truncated LdtMt2 (residues Leu131-Ala408) is reported in both ligand-free and meropenem-bound forms. The structure of meropenem-inhibited LdtMt2 provides a detailed structural view of the interactions between a carbapenem drug and Mtb L,D-transpeptidase. The structures revealed that the catalytic L,D-transpeptidase domain of LdtMt2 is preceded by a bacterial immunogloblin-like Big_5 domain and is followed by an extended C-terminal tail that interacts with both domains. Furthermore, it is shown using mass analyses that meropenem acts as a suicide inhibitor of LdtMt2. Upon acylation of the catalytic Cys354 by meropenem, the `active-site lid' undergoes a large conformational change to partially cover the active site so that the bound meropenem is accessible to the bulk solvent via three narrow paths. This work will facilitate structure-guided discovery of L,D-transpeptidase inhibitors as novel antituberculosis drugs against drug-resistant Mtb.

Keywords: LdtMt2; Mt2594; Mycobacterium tuberculosis; Rv2518c; antituberculosis drug discovery; carbapenem; l,d-transpeptidases; meropenem; peptidoglycans.

Figure 1

Overall structure of Ldt_Mt2Δ130. (a) Ribbon diagram of meropenem-complexed Ldt_Mt2Δ130. The NTD and the Ldt domain are shown in green and yellow, respectively. The active-site lid (His300–Asp323) and the C-terminal tail (Asn379–Ala408) are coloured red and blue, respectively. Meropenem bound to the Ldt domain is shown as a stick model. (b) Domains of Mtb Ldt_Mt2 coloured as in (a). TM, transmembrane helix. (c) Topology diagram of Ldt_Mt2Δ130 coloured as in (a). (d) Electrostatic surface diagram of meropenem-complexed Ldt_Mt2Δ130. Blue and red indicate positive and negative electrostatic potentials at neutral pH, respectively.

Figure 2

Structural comparisons and domain interactions in Ldt_Mt2Δ130. (a) A superposition of the four chains in the three Ldt_Mt2Δ130 models and a plot of the C^α r.m.s. deviations between any pair of chains averaged over the six pairwise comparisons. The apo, mercury-derivatized and meropenem-complexed Ldt_Mt2 (chains A and B) are coloured orange, magenta, yellow and blue, respectively. (b) Interactions between the C-terminal tail and two domains coloured as in Fig. 1 ▶(a). The enlarged views on the left have slightly different orientations in order to show the detailed interactions better.

Figure 3

Big_5 domain of Ldt_{c Mt2}. (a) Topology diagrams of the Big_5 domain (His150–Gly250) of Ldt_Mt2Δ130 (inset) and of four distinct subtypes of the Ig-like fold (modified from Bork et al., 1994 ▶): c-type (constant), v-type (variable), s-type (switched) and h-type (hybrid). The four-stranded structural core (strands b, c, e, and f; β2, β3, β5 and β6 of Ldt_Mt2Δ130) common to all Ig-like domains (orange) is surrounded by structurally more variable strands (green). (b, c) Ribbon diagrams of the Big_5 domain (His150–Gly250) in the apo model of Ldt_Mt2Δ130 (b) and meropenem-complexed Ldt_Mt2Δ130 (c), and enlarged views of the β6–β7 loop (insets) coloured as in (a). The bound calcium ion and the residues around it (Asp232–Met237) are shown as a purple ball and as stick models, respectively, with a 2mF _o − DF electron-density map (contoured at 1.5σ).

Figure 4

The active site of the Ldt domain and the substrate-binding sites. (a) Active-site superposition of the apo (orange), mercury-derivatized (magenta) and meropenem-complexed (chain B, cyan) Ldt_Mt2Δ130. Dotted lines denote interactions: hydrogen bonds to the catalytic triad (red) and the oxyanion hole (green), Ser351 with the oxyanion hole (black), His336 with Asn356 (black) and His352 with Cys354 S^γ and the main-chain carbonyl O atom of His352 in the mercury-derivatized model (purple). The covalently bound meropenem adduct in the meropenem complex is shown as a stick model. (b) Active-site superposition of meropenem-complexed Ldt_Mt2 (chain B, cyan), Ldt_Bs (green) and Ldt_fm (grey) in the same view as in (a). (c, d) Electrostatic surface representations of the predicted binding sites for the donor substrate in the open conformation of mercury-derivatized Ldt_Mt2Δ130 (c) and the acceptor substrate in the closed conformation of meropenem-complexed Ldt_Mt2Δ130 (d) coloured as in Fig. 1 ▶(d). The surfaces of the binding site are represented with constituent residues as stick models. The movement of the imidazole ring of His352 in the apo form (orange) and the mercury-derivatized form (magenta) is presented in stick models with dotted surfaces in (c). The peptide bond of the donor substrate (meso-DAP³-d-Ala⁴) is schematically modelled into the active site in the open conformation. The terminus of the acceptor substrate (meso-DAP³) is schematically modelled into the active site in the closed conformation, with red dotted lines depicting a plausible site for recognizing the terminal amine of meso-DAP³.

Figure 5

Conformational flexibility of the active-site lid in Ldt_Mt2Δ130. (a, b) Three different views of the electrostatic potential surface diagrams of mercury-derivatized (a) and meropenem-complexed Ldt_Mt2Δ130 (b). The closed conformation of the active-site lid in (b) reveals that meropem attached to catalytic Cys354 is accessible through three narrow paths (Paths A, B and C). (c) The active-site lids in meropenem-complexed (cyan) and mercury-derivatized (magenta) Ldt_Mt2Δ130 are shown as ribbon models with the surface of the meropenem complex. Residues that show large shifts upon lid closure are shown as stick models; the movement is indicated by black dotted arrows. The two models are in the same orientation.

Figure 6

Meropenem-inactivated Ldt_Mt2Δ130. (a) Electron-density map (left) and a schematic diagram of interactions (right) of the covalently bound meropenem adduct with Cys354 in meropenem-complexed Ldt_Mt2Δ130 (chain B). The OMIT mF _o − DF _c map (contoured at 2.5σ) for meropenem and the 2mF _o − DF _c map (contoured at 1.0σ) for Cys354 are coloured blue and yellow, respectively. Dotted lines denote interactions with Ldt_Mt2Δ130 and the corresponding bond lengths are shown in Å. Variable regions (R ¹, R ² and R ³) of carbapenems are shaded in green, blue and red, respectively. (b) Interactions of the bound meropenem with Tyr318 (left) and Tyr308 (right). Dotted lines and the electron-density map are presented as in (a). (c) Surface representation (left) and ribbon diagram (right) of the active site of Ldt_Mt2Δ130 enclosing the meropenem adduct viewed along Path B. The β14–β15 loop, the β15–β16 loop and the active-site lid, which surround the bound meropenem, are coloured plum, orange and green, respectively. (d) Surface representation (left) and ribbon diagram (right) of the active site in the meropenem-complexed Ldt_Mt2Δ130 viewed along Path A presented as in (c).