Atomic model of a cell-wall cross-linking enzyme in complex with an intact bacterial peptidoglycan

Abstract

The maintenance of bacterial cell shape and integrity is largely attributed to peptidoglycan, a highly cross-linked biopolymer. The transpeptidases that perform this cross-linking are important targets for antibiotics. Despite this biomedical importance, to date no structure of a protein in complex with an intact bacterial peptidoglycan has been resolved, primarily due to the large size and flexibility of peptidoglycan sacculi. Here we use solid-state NMR spectroscopy to derive for the first time an atomic model of an l,d-transpeptidase from Bacillus subtilis bound to its natural substrate, the intact B. subtilis peptidoglycan. Importantly, the model obtained from protein chemical shift perturbation data shows that both domains-the catalytic domain as well as the proposed peptidoglycan recognition domain-are important for the interaction and reveals a novel binding motif that involves residues outside of the classical enzymatic pocket. Experiments on mutants and truncated protein constructs independently confirm the binding site and the implication of both domains. Through measurements of dipolar-coupling derived order parameters of bond motion we show that protein binding reduces the flexibility of peptidoglycan. This first report of an atomic model of a protein-peptidoglycan complex paves the way for the design of new antibiotic drugs targeting l,d-transpeptidases. The strategy developed here can be extended to the study of a large variety of enzymes involved in peptidoglycan morphogenesis.

Figure 1

The impact of protein binding on peptidoglycan dynamics. (A) Chemical structure of peptidoglycan. (B) Measurement of one-bond ¹H-¹³C dipolar couplings in peptidoglycan, using a windowed R18₁⁷ sequence at a MAS frequency of 7.716 kHz. Dipolar splittings are measured in the absence (blue) and presence (red) of Ldt_Bs. Dotted lines show best-fit curves, based on numerical simulations of the pulse sequence (see Experimental section). (C) ¹H-¹³C dipolar couplings in peptidoglycan without (blue) and with (red) Ldt_Bs. (D) ¹³C T₁ relaxation time constants without (blue, data similar to ref. 19) and with (red) Ldt_Bs.

Figure 2

ssNMR characterization of the interaction between Ldt_Bs and B. subtilis peptidoglycan. A) Comparison of ¹H-¹⁵N correlation spectra of [U-²H,¹³C,¹⁵N] Ldt_Bs in solution (blue) and in the presence of peptidoglycan (red). The latter spectrum was collected at a MAS frequency of 39 kHz, using cross-polarization (CP) transfer steps. B) Representative excerpts from 3D ¹H-¹³C^α-¹⁵N correlation spectra. The peak labeled with an asterisk (lower right) arises from the correlation to the C^α of the preceding residue 164, which cannot be observed in the solid state due to pulse sequence design. Numbers in each panel refer to the ¹⁵N chemical shift at which the displayed ¹H-¹³C planes were extracted. C) Combined chemical shift perturbations (CSP) between free and bound protein, calculated as the square root of the sum of the squared absolute chemical shift difference in the ¹H,¹³C, ¹⁵N dimensions, weighted by the relative gyromagnetic ratios. Red arrows indicate the residues shown in panel (B). The red horizontal line displays two standard deviations over all residues.

Figure 3

NMR chemical-shift perturbation (CSP) induced by the peptidoglycan on Ldt_Bs and result of the HADDOCK calculation. A) CSP are displayed in red on a ribbon representation of Ldt_Bs. The threshold shown in Figure 2C was used in this representation. B) Lowest energy structure obtained for the peptidoglycan-Ldt_Bs complex. The catalytic cysteine (C142) is shown in yellow. The residues shown in blue are H122 (left panel) and V47 (right panel), used for mutation experiments.

Figure 4

Solution-state NMR performed on the individual LysM and catalytic domains. (A, B) 1D ¹H NMR spectra of the LysM domain recorded in the absence (A, blue) or presence (B, red) of peptidoglycan. C) Photographs of the respective samples described in A) and B). (D) ¹H-¹⁵N HSQC spectra recorded on these two LysM samples. Peaks corresponding to residues with significant chemical shift perturbation (CSP) upon peptidoglycan interaction are highlighted. Residue-wise CSP plots are shown in Figure S9. (E) Position of the backbone sites of residues with significant CSP are show as red spheres. The orientation of the LysM domain is identical to that used in Figure 3A (right panel). (F, G) 1D ¹H NMR spectra of the catalytic domain recorded in the absence (F, blue) or presence (G, red) of a peptidoglycan suspension. All 1D spectra resulted from 128 scans except for the spectrum in panel (G) for which 1024 scans were accumulated.

Figure 5

Possible localization of the peptidoglycan peptide stems into the catalytic pocket. A) Superimposition of our HADDOCK Ldt_Bs:peptidoglycan-hexamer :model (Ldt_{B s} cartoon structure in red and peptidoglycan in orange sticks) with the Ldt_fm acylenzyme structure (cyan cartoon and surface), with ertapenem (grey mesh). A peptide stem of peptidoglycan is localized in the same acyl donor pocket as the carbapenem antibiotic. B) Extension of our HADDOCK Ldt_Bs:peptidoglycan-hexamer model to a complete peptidoglycan polymer. The hexamer muropeptides of our model was overlapped with one of the glycosidic chain of the complete peptidoglycan polymer. The peptidoglycan was modeled using a threefold axis for the glycan chains. The peptide conformation was adapted to allow the cross-linking between adjacent glycan chains. Possible acceptor and donor peptide stems are represented in green and blue, respectively. The catalytic cysteine is shown in yellow.