Structure and Dynamics of Pentaglycyl Bridges in the Cell Walls of Staphylococcus aureus by ¹³C−¹⁵N REDOR NMR

Abstract

Whole cells and cell-wall fractions of Staphylococcusaureus have been labeled by various combinations of [1-13C]glycine, [15N]glycine, L--6-13C-lysine, L--6-15N-lysine, D--1-13C-alanine, and D--15N-alanine. The resulting materials have been examined using 13C and 15N solid-state, magic-angle spinning NMR techniques including cross-polarization, double cross-polarization, and rotational-echo double resonance. The results of these measurements indicate that the peptidoglycan glycyl bridges are complete (five units long) and form cross-links between three-quarters of all peptide stems. The pentaglycyl bridges are immobilized in lyophilized cell-wall fractions in a compact conformation with inter-residue spacings comparable to those of an alpha helix. The bridges have a similar compact conformation in intact whole cells, regardless of whether the cells have been lyophilized or were hydrated and frozen at -10 degrees C. The bridges are also in a time-averaged compact conformation in whole cells at 0 degrees C but with sizable structural fluctuations associated with local mobility. A small fraction of bridges are in extended-chain conformations.

Structures of Staphylococcus aureus Cell-Wall Complexes with Vancomycin, Eremomycin, and Chloroeremomycin Derivatives by 13C{19F} and 15N{19F} Rotational-Echo Double Resonance

“…Nevertheless, substantive differences are present between what we will call the final or "modified" [ 19 F] oritavancin model (Figures 15-16, top) and the MD model (30) of Cegelski et al (Figures 15-16, bottom). The differences are: (i) the pentaglycyl bridge in the modified model is helical, consistent with the reported glycyl carbonyl-carbon chemical shifts (15) and pentaglycyl endto-end distance (20); (ii) the pentaglycyl bridging segment is lowered into a protective cleft formed by the 4-epi-vancosamine and the glycopeptide core so that it will be in position to account for the 13 Figures 8 and 11); (iii) the D-iso-Gln of the bound stem is moved up towards the 4-epi-vancosamine moiety; and (iv) the unbound neighboring stem is moved away from the C-terminus of the glycopeptide core and proximity to the bound stem. In the discussion of other structural modifications that follows, reference to the [ 19 F]oritavancin-PG model will always be to the "modified" model of Figures 15-16 (top).…”

“…aureus cells were grown in 300 ml of defined media (20) containing [1][2][3][4][5][6][7][8][9][10][11][12][13] C]glycine and L-[ε -15 N]lysine. The cells were harvested at OD 660 = 1.0 and were complexed with 6.7 mg (4.0 μmoles) of FPBV, resulting in 66% cell-wall binding-site occupancy (15).…”

“…The observed asymptotic dephasing limit is 35%, in agreement with the calculated value. This is true despite the fact that LY309687 presumably binds to stems that have no pentaglycyl segment attached to the lysyl ε-nitrogen at Gly-5 (15% of all stems) (15), or to stems that are non-bridging and have no cross-link at Gly-1 (20% of all stems) (20), complexes for which the contribution to the dephasing limit would be reduced by one-third.…”

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

“…After introducing CPMAS in 1976, Schaefer was the first to implement solidstate NMR to examine whole-cell systems and bacterial cell walls, including studies with A. viridans [69], B. subtilis [70], S. aureus [50] and E. faecium [71], although the most extensive work to date has resulted from experiments with S. aureus.…”

Uniformity of Glycyl Bridge Lengths in the Mature Cell Walls of Fem Mutants of Methicillin-Resistant Staphylococcus aureus

“…The starter cultures were shaken at 200 rpm in an Environ-Shaker (Lab-Lines Instruments, Inc., Melrose Park, IL), maintained overnight at 37°C. The starter culture grown overnight (1% final volume) was added to 2 liters of sterile S. aureus standard medium (SASM) (25,27 13 C and 15 N labels in the bridging segment in the PG of intact cells (Fig. 1).…”