Oritavancin Binds to Isolated Protoplast Membranes but not Intact Protoplasts of Staphylococcus aureus

Journal of Molecular Biology Volume 391, Issue 2, 14 August 2009, Pages 414-425

Solid-state NMR has been used to examine the binding of N'-4-[(4-fluorophenyl)benzyl)]chloroeremomycin, a fluorinated analogue of oritavancin, to isolated protoplast membranes and whole-cell sucrose-stabilized protoplasts of Staphylococcus aureus, grown in media containing [1-13C]glycine and l-[var epsilon-15N]lysine. Rotational-echo double-resonance NMR was used to characterize the binding by estimating internuclear distances from 19F of oritavancin to 13C and 15N labels of the membrane-associated peptidoglycan and to the 31P of the phospholipid bilayer of the membrane. In isolated protoplast membranes, both with and without 1 M sucrose added to the buffer, the nascent peptidoglycan was extended away from the membrane surface and the oritavancin hydrophobic side chain was buried deep in the exposed lipid bilayer. However, there was no N'-4-[(4-fluorophenyl)benzyl)]chloroeremomycin binding to intact sucrose-stabilized protoplasts, even though the drug bound normally to the cell walls of whole cells of S. aureus in the presence of 1 M sucrose. As shown by the proximity of peptidoglycan-bridge 13C labels to phosphate 31P, the nascent peptidoglycan of the intact protoplasts was confined to the membrane surface.

Chemical structures of vancomycin and oritavancin.

Transmission electron micrographs of whole-cell protoplasts and isolated protoplast membranes prepared from S. aureus. (Left) Protoplasts prepared by incubating S. aureus in 75 ug/mL of lysostaphin for 1 h. The protoplast conversion was uniform with no visible parent forms. A lysed protoplast with a hollow membrane shell is marked by an asterisk. (Right) Isolated protoplast membranes prepared from whole-cell protoplasts. Only membrane vesicles varying in size with diameters ranging from 0.05 to 0.8 um are visible. Mesosomal vesicles have diameters less than 0.1 um.

13C{19F} REDOR spectra (125 MHz) after 8.96 ms of dipolar evolution of [19F]oritavancin complexed to intact protoplasts (left) and isolated protoplast membranes (right) prepared from S. aureus labeled by [1-13C]glycine. The full-echo spectra (S0) are at the bottom of the figure, and the REDOR-difference spectra (DS = S0 - S, where S is the dephased-echo spectrum) are shown at the top. The S0 spectra were normalized for equal intensity of the 171-ppm peak. The spectra on the left were the result of the accumulation of 38,948 scans, and the spectra on the right were the result of the accumulation of 21,112 scans. MAS was at 7143 Hz.

Illustrations of possible nascent peptidoglycan organization in intact protoplasts (left) and isolated protoplast membranes (right) consistent with the REDOR results. (Top) The gray circle represents the lipid bilayer of the cytoplasmic membrane. The black line represents the disaccharide of a peptidoglycan repeat unit, the green line represents the stem structure, and the red line represents the bridge structure. Equal quantities of lipid II and nascent peptidoglycan are shown in both intact protoplasts and isolated protoplast membranes. The nascent peptidoglycan strands are parallel with the membrane surface in intact protoplasts and perpendicular in isolated protoplast membranes. Close-up views of the nascent peptidoglycan organization are shown in the bottom panels of the figure. Color coding of the peptidoglycan units is the same in all panels. In the isolated protoplast membranes, [19F]oritavancin (blue) is shown with its hydrophobic side chain embedded within the membrane and the fluorine positioned near the middle of the lipid bilayer.