The Carboxyl Terminus of Eremomycin Facilitates Binding to the Non-D-Ala-D-Ala Segment of the Peptidoglycan Pentapeptide Stem

Glycopeptide antibiotics inhibit cell wall biosynthesis in Gram-positive bacteria by targeting the peptidoglycan (PG) pentapeptide stem structure (L-Ala-D-iso-Gln-L-Lys-D-Ala-D-Ala). Structures of the glycopeptide complexed with a PG stem mimic have shown that the D-Ala-D-Ala segment is the primary drug binding site; however, biochemical evidence suggests that the glycopeptide-PG interaction involves more than D-Ala-D-Ala binding. Interactions of the glycopeptide with the non-D-Ala-D-Ala segment of the PG stem were investigated using solid-state nuclear magnetic resonance (NMR). LCTA-1421, a double N-15-enriched eremomycin derivative with a C-terminal [N-15]amide and [N-15]Asn amide, was complexed with whole cells of Staphylococcus aureus grown in a defined medium containing L-[3-C-13]Ala and D-[1-C-13]Ala in the presence of alanine racemase inhibitor alaphosphin. C-13{N-15} and N-15{C-13} rotational-echo double-resonance (REDOR) NMR measurements determined the C-13-N-15 internuclear distances between the [N-15]Asn amide of LCTA-1421 and the C-13 atoms of the bound D-[1-C-13]Ala-D-[1-C-13]Ala to be 5.1 and 4.8 angstrom, respectively. These measurements also determined the distance from the C-terminal [N-15]amide of LCTA-1421 to the L-[3-C-13]Ala of PG to be 3.5 angstrom. The measured REDOR distance constraints position the C-terminus of the glycopeptide in the proximity of the L-Ala of the PG, suggesting that the C-terminus of the glycopeptide interacts near the L-Ala segment of the PG stem. In vivo REDOR measurements provided structural insight into how C-terminally modified glycopeptide antibiotics operate.