Three-dimensional structure of the bacterial cell wall peptidoglycan

Abstract

The 3D structure of the bacterial peptidoglycan, the major constituent of the cell wall, is one of the most important, yet still unsolved, structural problems in biochemistry. The peptidoglycan comprises alternating N-acetylglucosamine (NAG) and N-acetylmuramic disaccharide (NAM) saccharides, the latter of which has a peptide stem. Adjacent peptide stems are cross-linked by the transpeptidase enzymes of cell wall biosynthesis to provide the cell wall polymer with the structural integrity required by the bacterium. The cell wall and its biosynthetic enzymes are targets of antibiotics. The 3D structure of the cell wall has been elusive because of its complexity and the lack of pure samples. Herein we report the 3D solution structure as determined by NMR of the 2-kDa NAG-NAM(pentapeptide)-NAG-NAM(pentapeptide) synthetic fragment of the cell wall. The glycan backbone of this peptidoglycan forms a right-handed helix with a periodicity of three for the NAG-NAM repeat (per turn of the helix). The first two amino acids of the pentapeptide adopt a limited number of conformations. Based on this structure a model for the bacterial cell wall is proposed.

Fig. 1.

Chemical structure and solution conformation of the peptidoglycan segment. (A) Chemical structure of compound 1. (B) NMR structure of the synthetic segment 1 of the cell wall, shown as a stereoview of 20 superimposed conformers. The disordered segments of the peptide stems (the l-Lys-d-Ala-d-Ala termini) are omitted for clarity. The conformers are depicted in capped sticks representation with O atoms in red, N atoms in blue, and C atoms in yellow.

Fig. 2.

The ¹³C heteronuclear sequential quantum correlation spectrum of 1 showing ¹³C correlations for the ¹H resonances between δ 2.8 and 5.0. This 600-MHz (with respect ¹H) spectrum was obtained at 298 K by using a 15-mM solution of 1 in 100% D₂O. Spectral features folded in the carbon dimension are identified by ∗.

Fig. 3.

The [NAG-NAM]₈ octamer construct derived from the NMR structure of 1. (A) Stereoview of a representative [NAG-NAM]₈ conformer, shown in capped-stick representation (O, N, and C are shown in red, blue, and yellow, respectively). (B) Solvent-accessible Connolly surface representation with the glycan backbone shown in orange and the peptide in green for the same perspective shown in A. (C) Stereoview of this construct seen from above (down the helical axis), shown as a solvent-accessible Connolly surface with the same the color coding as in B.

Fig. 4.

Structure of the polymeric peptidoglycan segment. (A) Schematic of the top view of the structure of the cell wall with each peptidoglycan strand represented as a three-pronged star, mimicking the image shown in B. (B and C) Top (B) and side (C) views of the extended model of the structure of the cell wall. A Connolly solvent-accessible surface is used, showing the glycan strands in orange and the peptide stems in green (as also depicted in Fig. 3B for an individual strand of peptidoglycan). The top view is shown to a depth of eight NAG-NAM repeats, and double-headed arrows span a small and a larger pore. The longest strands in the side view are also eight NAG-NAM repeats. For reference, as determined by Matias et al. (42) the approximate thickness of the cell wall components for E. coli are: plasma membrane, 58 Å; peptidoglycan, 64 Å; periplasm, 210 Å; outer membrane (having a truncated lipopolysaccharide), 69 Å.

Fig. 5.

Stereoviews from the side of (A) and above (B) the crystal structure of TolC outer-membrane channel embedded into a pore of the cell wall depicted in Fig. 4. The TolC protein is shown in ribbon and surface representations by using a purple solvent-accessible Connolly surface. The protruding TolC β-barrel domain (shown in ribbon) is fully embedded into the outer-membrane bilayer of the Gram-negative bacterium. As the peptidoglycan is in direct contact with the inner leaflet of the outer membrane bilayer, the juxtaposition of the TolC within the peptidoglycan is reasonable. The glycan chains are eight NAG-NAM repeats in length.