Cross-linking in the Living Cell Locates the Site of Action of Oxazolidinone Antibiotics

• Published in: The Journal of biological chemistry Vol. 278; no. 24; pp. 21972 - 21979
• Main Authors: Colca, Jerry R., McDonald, William G., Waldon, Daniel J., Thomasco, Lisa M., Gadwood, Robert C., Lund, Eric T., Cavey, Gregory S., Mathews, W. Rodney, Adams, Lonnie D., Cecil, Eric T., Pearson, James D., Bock, Jeffrey H., Mott, John E., Shinabarger, Dean L., Xiong, Liqun, Mankin, Alexander S.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 13.06.2003; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Anti-Bacterial Agents - pharmacology; Binding Sites; Cross-Linking Reagents - pharmacology; Electrophoresis, Polyacrylamide Gel; Models, Chemical; Models, Genetic; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Oxazolidinones - pharmacology; Peptides - chemistry; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Protein Synthesis Inhibitors - pharmacology; RNA - metabolism; RNA, Ribosomal, 23S - metabolism; RNA, Transfer - metabolism; Staphylococcus aureus - metabolism; Transcription Factors - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M302109200

Oxazolidinone antibiotics, an important new class of synthetic antibacterials, inhibit protein synthesis by interfering with ribosomal function. The exact site and mechanism of oxazolidinone action has not been elucidated. Although genetic data pointed to the ribosomal peptidyltransferase as the primary site of drug action, some biochemical studies conducted in vitro suggested interaction with different regions of the ribosome. These inconsistent observations obtained in vivo and in vitro have complicated the understanding of oxazolidinone action. To localize the site of oxazolidinone action in the living cell, we have cross-linked a photoactive drug analog to its target in intact, actively growing Staphylococcus aureus. The oxazolidinone cross-linked specifically to 23 S rRNA, tRNA, and two polypeptides. The site of cross-linking to 23 S rRNA was mapped to the universally conserved A-2602. Polypeptides cross-linked were the ribosomal protein L27, whose N terminus may reach the peptidyltransferase center, and LepA, a protein homologous to translation factors. Only ribosome-associated LepA, but not free protein, was cross-linked, indicating that LepA was cross-linked by the ribosome-bound antibiotic. The evidence suggests that a specific oxazolidinone binding site is formed in the translating ribosome in the immediate vicinity of the peptidyltransferase center.