Rotational-Echo Double Resonance Characterization of Vancomycin Binding Sites in Staphylococcus aureus

Abstract

Solid-state NMR experiments with stable isotope-labeled Staphylococcus aureus have provided insight into the structure of the peptidoglycan binding site of a potent fluorobiphenyl derivative of chloroeremomycin (Eli Lilly LY329332). Rotational-echo double resonance (REDOR) NMR provided internuclear distances from the 19F of this glycopeptide antibiotic to natural-abundance 31P and to specific 13C and 15N labels biosynthetically incorporated into the bacteria from labeled alanine, glycine, or lysine in the growth medium. Results from experiments with intact late log phase bacteria and cell walls indicated homogeneous drug-peptidoglycan binding. Drug dimers were not detected in situ, and the hydrophobic fluorobiphenyl group of LY329332 did not insert into the bilayer membrane. A model of the binding site consistent with the REDOR results positions the vancomycin cleft around an un-cross-linked D-Ala-D-Ala peptide stem with the fluorobiphenyl moiety of the antibiotic near the base of a second, proximate stem in a locally ordered peptidoglycan matrix.

Getting Closer to the Real Bacterial Cell Wall Target: Biomolecular Interactions of Water‐Soluble Lipid II with Glycopeptide Antibiotics

“…In fact Kim et al [42] have recently demonstrated by rotational-echo double resonance of vancomycin binding sites in Staphylococcus aureus that glycopeptide antibiotics might not form dimers in-situ in mature peptidoglycan. Furthermore, they postulate that the sugar interactions with the glycans of the cell wall stabilize the monomeric complex.…”

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

“…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). A second sample contained 6.7 mg LY309687 (3.7 μmol) complexed with whole cells for 60% binding-site occupancy.…”

“…The calculated dephasing is from five independent 13 C-19 F pairs (15). The weak 13 C- 13 C coupling between carbonyl carbons of the pentaglycyl bridging segment is totally suppressed by fast magic-angle spinning, and the 3-bond J coupling is less than 10 Hz.…”

“…This limit can be calculated from the amount of complexed drug, known from the binding assay (15), and the fraction of crosslinked stems in the cell walls of S. aureus, directly determined by a transferred-echo doubleresonance (TEDOR) experiment (30). Each pentaglycyl bridge is attached at its Gly A binding assay (15) showed that 2/3 of all binding sites are occupied by FPBV. Thus, the fraction of all pentaglycyl segments that are near a fluorine in the FPBV-PG complex is (2/3) (.46) = 31%, which is the calculated dephasing limit.…”

“…The experiments were similar to those performed earlier on [ 19 F]oritavancin-PG complexes (15). REDOR was used to measure the dipolar couplings between 19 F of the drugs, and 13 C and 15 N labels incorporated in peptidoglycan (PG) stems and bridging pentaglycyl segments.…”