Loss of a Functionally and Structurally Distinct ld-Transpeptidase, LdtMt5, Compromises Cell Wall Integrity in Mycobacterium tuberculosis

Abstract

The final step of peptidoglycan (PG) biosynthesis in bacteria involves cross-linking of peptide side chains. This step in Mycobacterium tuberculosis is catalyzed by ld- and dd-transpeptidases that generate 3→3 and 4→3 transpeptide linkages, respectively. M. tuberculosis PG is predominantly 3→3 cross-linked, and LdtMt2 is the dominant ld-transpeptidase. There are four additional sequence paralogs of LdtMt2 encoded by the genome of this pathogen, and the reason for this apparent redundancy is unknown. Here, we studied one of the paralogs, LdtMt5, and found it to be structurally and functionally distinct. The structures of apo-LdtMt5 and its meropenem adduct presented here demonstrate that, despite overall architectural similarity to LdtMt2, the LdtMt5 active site has marked differences. The presence of a structurally divergent catalytic site and a proline-rich C-terminal subdomain suggest that this protein may have a distinct role in PG metabolism, perhaps involving other cell wall-anchored proteins. Furthermore, M. tuberculosis lacking a functional copy of LdtMt5 displayed aberrant growth and was more susceptible to killing by crystal violet, osmotic shock, and select carbapenem antibiotics. Therefore, we conclude that LdtMt5 is not a functionally redundant ld-transpeptidase, but rather it serves a unique and important role in maintaining the integrity of the M. tuberculosis cell wall.

Keywords: Mycobacterium tuberculosis; antibiotics; biosynthesis; cell wall; enzyme kinetics; enzyme structure; peptidoglycan.

FIGURE 1.

Crystal structure of Ldt_Mt5. Left, apo-Ldt_Mt5; right, rotated 90°. Ldt_Mt5 is composed of two BIg domains and a CD. The semitransparent volume is surface-accessible by a 3.5-Å radius probe. Residues of the active site (His³⁴², Thr³⁵⁷, Asn³⁵⁸, and Cys³⁶⁰) are represented as sticks within the CD. Tryptophan residues of the CTSD are also represented as sticks and interact with a hydrophobic patch at the interface of the BIgB domain. The prominent outer cavity that is observed in Ldt_Mt2 is absent in apo-Ldt_Mt5 but is indicated as a reference (10). The Ldt_Mt5 secondary structure schematic is colored as a rainbow from blue (N terminus) to red (C-terminus). Orange dashes represent the disordered portion of loop L_C, and red dashes represent the disordered ex-CTSD. This figure was made using Chimera (19).

FIGURE 2.

Comparison of M. tuberculosisld-transpeptidases. A, the sequence alignment based on the structural superposition. The observed secondary structures are noted above the amino acid sequences. Starred residues are those highlighted in the text (blue, coordinating His³⁴²; cyan, BIgA and BIgB interface; green, CTSD-BIgB-CD core interaction; red, catalytic residues; yellow, loop L_D). Named loops are also marked, and the characteristic ld-transpeptidase motif is boxed in blue rectangle. B, overlay of the apo structures of Ldt_Mt2 (Protein Data Bank code 3VYN; in cyan) and Ldt_Mt5 (this work; in red). Observed differences in the CD are concentrated in the β-hairpin and loops L_C–L_E. Disordered regions are represented as dashed lines. C, the Ldt_Mt5 adduct structure displays the largest CD conformational changes among characterized ld-transpeptidases. The EYY-folded CD cores of apo-Ldt_Mt5 (red), holo-Ldt_Mt5 (pink), and the apo and holo crystal structures of Ldt_Mt1 (Protein Data Bank code 4JMN, apo, in light green; Protein Data Bank code 4JMX, imipenem adduct, in dark green), and Ldt_Mt2 (Protein Data Bank code 3VYN, apo, in cyan; Protein Data Bank code 3VYP, meropenem adduct, in blue) are shown as smoothed Cα traces. Structural elements with high r.m.s.d. are shown in full opacity; the remaining structural elements are displayed as transparent Cα traces to demonstrate conservation of the EYY-folded CD cores. The largest changes among structurally characterized ld-transpeptidases are observed in the β-hairpin, and in the case of Ldt_Mt5, there is a dramatic displacement of loop L_C that occurs after adduct formation (indicated with red curved arrows). The C-terminal portion of the CTSDs was excluded for clarity. D, accessible surface map of apo-Ldt_Mt5 colored by the magnitude of the observed atomic temperature factors from low (green) to high (magenta) motility. The flexibility of the β-hairpin as indicated by the high atomic temperature factor correlates with its large displacement upon adduct formation. These images, the sequence alignment, and structural superpositions were performed using the program MOE. The sequence representation was performed using ESPript3 (48).

FIGURE 3.

Comparison of the Ldt_Mt5 and Ldt_Mt2 active sites. A, apo-Ldt_Mt5; B, meropenem-bound Ldt_Mt5 (meropenem adduct shown as purple sticks); C, Ldt_Mt2-PG fragment complex (Protein Data Bank code 3TUR; PG fragment shown as cyan sticks). The left panels show secondary structure schematic representations, and residues within the active site are represented as sticks. The right panels show the probe-accessible surface (3.5-Å radius). Surface zones related to the β-hairpin and loop L_C that display the largest structural differences among apo and holo structures are colored purple. Acylation of Ldt_Mt5 by meropenem causes displacements of these structural elements as indicated by the green arrows (right panel) that “restore” the outer cavity.

FIGURE 4.

An “active” conformation of Ldt_Mt5 is achieved when meropenem binds. A, a nonproductive orientation of His³⁴² is observed in apo-Ldt_Mt5 (σA-weighted electron density map contoured at 1.0 σ level). Residues of the refined structures are shown as stick representations. His³⁴² is in close proximity to Cys³⁶⁰ as suggested by the electron density connecting these two residues. In addition, His³⁴² hydrogen bonds with the most populated alternative conformation of Glu³²⁸ and Asn³⁶² (dashed lines). Collectively, these interactions fix the imidazole ring in a nonproductive orientation that is not present in the meropenem adduct structure. B, mechanism of acylation of Ldt_Mt5 by meropenem. C, overlay of active sites of apo and meropenem adduct structures. The red arrows indicate the large displacements of Met³⁴⁶ (Sδ–Sδ, 6.6 Å), Thr³⁵⁷ (Cβ–Cβ, 8.2 Å), and Asn³⁵⁸ (Cβ–Cβ, 4.1 Å) relative to the apo structure. The active site of apo-Ldt_Mt5 is colored gray, and the meropenem adduct structure is colored brown. The ∼180° rotation of His³⁴² is noted with a curved red arrow. Meropenem is represented as magenta sticks. Disordered residues are represented as dashed lines. This figure was made using Chimera (19).

FIGURE 5.

pH rate profile analysis of Ldt_Mt5 (open circles) and Ldt_Mt2 (closed circles). Error bars represent S.E.

FIGURE 6.

Ldt_Mt5 active site variants catalyze nitrocefin hydrolysis at pH 10. Michaelis-Menten curves for Ldt_Mt5 and Ldt_Mt5 variants are shown. Error bars represent S.E.

FIGURE 7.

Loss of Ldt_Mt5 sensitizes M. tuberculosis to crystal violet and osmotic stress. Wild-type (closed circles), ldt_Mt5::Tn (open circles), or ldt_Mt2::Tn (triangles) M. tuberculosis were grown in 7H9 complete medium (A) or 7H9 medium supplemented with crystal violet (B). C, strains lacking Ldt_Mt2 or Ldt_Mt5 are less tolerant to osmotic shock. Error bars represent S.E.

FIGURE 8.

Transmission (A and C) and scanning (B and D) electron microscopy reveals no significant changes in M. tuberculosis cell morphology upon loss of a functional copy of Ldt_Mt5. A and B, wild-type M. tuberculosis; C and D, ldt_Mt5::Tn M. tuberculosis. Scale bars for transmission EM images (A and C) and scanning EM images (B and C) represent 100 and 200 nm, respectively.

FIGURE 9.

Loss of Ldt_Mt2 or Ldt_Mt5 compromises M. tuberculosis cell wall integrity. The M. tuberculosis PG is 3→3 and 4→3 cross-linked by ld- and dd-transpeptidases (not shown), respectively. Both Ldt_Mt2 (green) and Ldt_Mt5 (blue) have two BIg domains, whereas Ldt_Mt1 (red) has one, which likely differentially positions the ld-transpeptidases within the periplasm. Although loss of Ldt_Mt1 alone results in no discernible phenotype, loss of Ldt_Mt2 results in compromised cell wall integrity as a result of loss of 3→3 cross-links in PG. Ldt_Mt5 has a structurally distinct active site relative to both Ldt_Mt1 and Ldt_Mt2, and when Ldt_Mt5 is lost, M. tuberculosis is sensitized to chemical probes and osmotic stress similarly to when Ldt_Mt2 is lost. Although Ldt_Mt5 can catalyze 3→3 cross-link formation in vitro, its physiological function remains unclear.