BIOPHYSICS SEMINAR<br>Cell-Wall Architecture of Staphylococcus Aureus and the Mode of Action of Glycopeptide Antibiotics by Solid-State NMR

The chemical structure of the Staphylococcus aureus peptidoglycan (PG) repeat unit consists of a disaccharide, a pentapeptide stem, and a bridge connecting adjacent stems.  The pentapeptide stem has the amino acid sequence, l-alanine-d-iso-glutamine-l-lysine-d-alanine-d-alanine, and the bridge is a pentaglycyl segment.  PG units form a cross-linked three-dimensional cell-wall lattice by the cooperative action of a transglycosylase-transpeptidase.  While the chemical structure of the PG repeat unit is known, the three-dimensional structure of the PG lattice is unknown.

We have examined the PG tertiary structure in intact whole-cells of S. aureus using solid-state NMR.  The PG was selectively labeled by growing bacteria in the presence of an alanine racemase inhibitor (alaphosphin) in defined media containing d-[1-¹³C]alanine and l-[¹⁵N]alanine.  Rotational-echo double-resonance (REDOR) NMR was used to determine heteronuclear dipolar coupling, hence internuclear distances, between the ¹³C and ¹⁵N labels in the PG stems, and from these labels to the ¹⁹F of a glycopeptide drug bound specifically to the PG lattice.  These REDOR distance constraints are consistent with a model for local order in a repeating structural motif from which the PG lattice of S. aureus is assembled such that membrane-bound protein complexes and other large functional units are accomodated.  Within this motif, the glycan chains (repeating disaccharides) (i) have a 4-fold screw-axis helical conformation with a 40-Å periodicity, (ii) have cross-linked stems that are parallel to one another, and (iii) are themselves proximate to the cross-linking bridges.  Glycopeptide drugs that bind to nascent PG (incompletely cross-linked PG that is just entering the lattice) interfere with the ordering of the structural motif and are therefore potent antibiotics.