Staphylococcus aureus Peptidoglycan Tertiary Structure from Carbon-13 Spin Diffusion

J. Am. Chem. Soc., 2009, 131 (20), pp 7023--7030

The cell-wall peptidoglycan of Staphylococcus aureus is a heterogeneous, highly cross-linked polymer of unknown tertiary structure. We have partially characterized this structure by measuring spin diffusion from 13C labels in pentaglycyl cross-linking segments to natural-abundance 13C in the surrounding intact cell walls. The measurements were performed using a version of centerband-only detection of exchange (CODEX). The cell walls were isolated from S. aureus grown in media containing [1-13C]glycine. The CODEX spin diffusion rates established that the pentaglycyl bridge of one peptidoglycan repeat unit of S. aureus is within 5 A of the glycan chain of another repeat unit. This surprising proximity is interpreted in terms of a model for the peptidoglycan lattice in which all peptide stems in a plane perpendicular to the glycan mainchain are parallel to one another.

(a) Chemical Structure of the Peptidoglycan of Staphylococcus aureus and Two of Its Mutants. (b) Expansion of the Cross-Link to Bridge-Link Structure. The partial stem on the left contains a d-Ala-Gly cross-link (peptide bond), and the full stem on the right, a Gly-l-Lys (isopeptide bond) bridge-link. The bridging glycyl segment is attached to the l-Lys of the stem on the right, which ends in a d-Ala-d-Ala vancomycin binding site. The glycine content in the side chain differs from n = 5 (S. aureus strains BB255 and mutant Mu50) to n = 1 (FemA).

Pulse sequence for measurement of 13C spin diffusion by CODEX. All times are multiples of the rotor period, tr. When tm occurs between the first pair of store-and-read 90-degree pulses and tz between the second pair (as illustrated), the acquired signal is S. When the positions of tm and tz are interchanged, the acquired signal is S0. The sum of tm and tz is a constant.

CODEX spectra of intact cell walls of S. aureus strain BB255 (labeled by 5%-enriched [1-13C]glycine) shown on the left, and on the right, of mutant strain FemA (labeled by 99%-enriched [1-13C]glycine), after a mixing time, tm = 840 ms. The CODEX differences (DS = S0 - S) are at the top of the figure, and the CODEX references (S0), at the bottom. Normalization is the same as in Figure 2. The spectra on the left are the result of the accumulation of 245632 scans, and on the right, 166272 scans. Magic-angle spinning was at 7143 Hz.

Space-filling models of the cross sections of peptidoglycan lattice. Four parallel stems (green) are shown on the left, and six on the right. The d-alanine residues of the stems are in light blue, the pentaglycyl bridges in pink, the carbonyl carbons of the bridges in red, the glycan sugars in gray, and the anomeric carbons of the sugars in blue. The glycan mainchains are propagating into the plane of the paper. The anomeric sugar carbons are proximate to the glycyl carbonyl carbons in the cross-section on the left, but not in the cross-section on the right.