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

Abstract

Solid-state NMR has been used to examine isolated cell walls and intact whole cells of Staphylococcus aureus complexed to 5 different vancomycin, eremomycin, and chloroeremomycin derivatives. The cell walls and whole cells were specifically labeled with D-[1-13 C]alanine, or a combination of [1-13 C]glycine and [ε-15 N]lysine. Each of the bound glycopeptides had a 19 F-labeled substituent at either its C-terminus or disaccharide position. The 13 C{ 19 F} rotational-echo doubleresonance (REDOR) dephasing for the cell-wall 13 C-labeled bridging pentaglycyl segment connecting a glycopeptide-complexed peptidoglycan stem with its neighboring stem indicates that the fluorine labels for all bound glycopeptides are positioned at one end or the other of the bridge. An exception is N'-(p-trifluoromethoxybenzyl)chloroeremomycin whose hydrophobic substituent differs in length by one phenyl group compared to that of oritavancin, N'-4- [(4-chlorophenyl) benzyl)]chloroeremomycin. For this drug the fluorine label is near the middle of the pentaglycyl segment. The 15 N{ 19 F} REDOR dephasing shows proximity of the fluorine to the bridge-link site of the pentaglycyl bridge for C-terminus-substituted moieties, and to the cross-link site for disaccharide-substituted moieties. Full-echo REDOR spectra of cell-wall complexes from cells labeled by D-[1-13 C]alanine (in the presence of an alanine racemase inhibitor) reveal three different carbonyl-carbon chemical-shift environments, arising from the D-Ala-D-Ala binding site and the DAla-Gly-1 cross-link site. The REDOR results indicate a single fluorine dephasing center in each peptidoglycan complex. Molecular models of the mature cell-wall complexes that are consistent with internuclear distances obtained from 13 C{ 19 F} and 15 N{ 19 F} REDOR dephasing allow a correlation of structure and antimicrobial activity of the glycopeptides. KeywordsDipolar coupling; glycopeptide antibiotic; magic-angle spinning; peptidoglycan; solid-state NMR; transglycosylase Vancomycin is a potent antibiotic that is effective against multi-drug-resistant Gram-positive bacteria including methicillin-resistant S. aureus. As many as 60% of clinically isolated strains of S. aureus are methicillin resistant (1), which means that vancomycin is one of the most important antibiotics in use today. Vancomycin inhibits the transglycosylation step in the † This paper is based on work supported by the National Institutes of Health under grant number EB02058. * Jacob Schaefer, NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 August 9. The emergence of vancomycin-resistant enterococci (VRE) has limited vancomycin usage against methicillin-resistant S. aureus. In 2002, vancomycin-resistant S. aureus (VRSA) with a minimum inhibitory concentration (MIC) of greater than 128 μg/mL was recovered from a patient in Michigan who was being treated with multiple antibiotics (5). E. faecalis, a vancomycin-resistant enterococcus, was also recovered from the patient. The VRSA isolat...

Structural Variations of the Cell Wall Precursor Lipid II and Their Influence on Binding and Activity of the Lipoglycopeptide Antibiotic Oritavancin

“…Here, we provide evidence at the molecular level that these structural modifications enable oritavancin, in contrast to vancomycin, to bind to further target sites of the lipid II peptidoglycan precursor other than the D-Ala-D-Ala terminus. With use of solidstate nuclear magnetic resonance (NMR) and rotational-echo double resonance (REDOR), a model for the oritavancin-PG complex has been proposed in which the crossbridge is bound in a cleft between the epi-vancosamine residue and the core of the oritavancin molecule (36,39,40). We now provide biochemical evidence that the crossbridge and the D-iso-glutamine in position 2 of the lipid II stem peptide represent crucial binding sites for oritavancin.…”

“…In VRSA, the terminal D-Ala residue is exchanged to D-Lac, and it has been proposed that oritavancin compensates for the resulting loss of one critical hydrogen bond to this target through the interaction with additional binding sites on the lipid II molecule, i.e., the crossbridge (36,39). However, vanA-type vancomycinresistant S. aureus strains are characterized by a complete lack of or an incomplete chain length of the pentaglycine crossbridge (41), suggesting that in these strains yet another binding site on the lipid II molecule contributes to the high-affinity binding.…”

“…Recent studies suggested that binding of oritavancin involves multiple interactions with its molecular target lipid II. Solid-state nuclear magnetic resonance (NMR) and computational modeling predicted secondary interactions of the 4=-chlorobiphenyl methyl side chain with the S. aureus pentaglycine crossbridge and the D-aspartate/D-asparagine (D-Asx) crossbridge in Enterococcus faecium (36)(37)(38)(39)(40). To better understand the subtleties of the mechanism of action of oritavancin, we characterized the binding to its target lipid II in vivo and in vitro using purified lipid II variants.…”

Live-cell high resolution magic angle spinning magnetic resonance spectroscopy for in vivo analysis of Pseudomonas aeruginosa metabolomics

“…Within our analysis, we detected signals corresponding to cell wall polysaccharides derived from N-acetyl termini (2.02÷2.33/4.10÷4.33 ppm) (27). This is in accordance with our previous results (28) which demonstrated that the PA cell wall contains peptidoglycans, and is a thick rigid layer composed of an overlapping lattice of the sugars, N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM), cross-linked by amino acid bridges (2003)(2004)(2005)(2006), The Microbical World http://www.microbiologytext.com).…”

Inhibition of Staphylococcus aureus Cell Wall Biosynthesis by Desleucyl-Oritavancin: a Quantitative Peptidoglycan Composition Analysis by Mass Spectrometry

“…Since oritavancin readily forms dimers in solution and binds to membrane vesicles (8), drug dimerization and membrane anchoring mediated by the hydrophobic side chain of oritavancin are thought to increase its binding to the D-Ala-D-Lac-terminated PG stem structure to overcome the vancomycin resistance in VRE (9). However, in situ characterizations of oritavancin (10) and oritavancin-like glycopeptide (4,11,12) binding sites in intact whole cells of S. aureus by solid-state nuclear magnetic resonance (NMR) have shown that lipoglycopeptides do not bind to the cell wall as dimers or partition to the bacterial membrane. The only instance of oritavancin's hydrophobic side chain being found inserted into the lipid bilayer was when it was complexed to an isolated protoplast membrane of S. aureus.…”