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Mutagenesis mapping of the protein-protein interaction underlying FusB-type fusidic acid resistance
Georgina Cox1, Thomas A Edwards, Alex J O'Neill
1Antimicrobial Research Centre and School of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom.
Abstract:
FusB-type proteins represent the predominant mechanism of resistance to fusidic acid in staphylococci and act by binding to and modulating the function of the drug target (elongation factor G [EF-G]). To gain further insight into this antibiotic resistance mechanism, we sought to identify residues important for the interaction of FusB with EF-G and thereby delineate the binding interface within the FusB-EF-G complex. Replacement with alanine of any one of four conserved residues within the C-terminal domain of FusB (F156, K184, Y187, and F208) abrogated the ability of the protein to confer resistance to fusidic acid; the purified mutant proteins also lost the ability to bind S. aureus EF-G in vitro. E. coli EF-G, which is not ordinarily able to bind FusB-type proteins, was rendered competent for binding to FusB following deletion of a 3-residue tract (529SNP531) from domain IV of the protein. This study has identified key regions of both FusB and EF-G that are important for the interaction between the proteins, findings which corroborate our previous in silico prediction for the architecture of the complex formed between the resistance protein and the drug target (G. Cox, G. S. Thompson, H. T. Jenkins, F. Peske, A. Savelsbergh, M. V. Rodnina, W. Wintermeyer, S. W. Homans, T. A. Edwards, and A. J. O'Neill, Proc. Natl. Acad. Sci. U. S. A. 109:2102-2107, 2012).
Insights
FusB proteins confer fusidic acid resistance by binding elongation factor G (EF-G). Key residues in FusB and EF-G were identified, clarifying the interaction mechanism crucial for antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Drug Resistance
Background:
- FusB-type proteins are the primary mechanism for fusidic acid resistance in staphylococci.
- These proteins function by binding to and altering the activity of the drug target, elongation factor G (EF-G).
Purpose of the Study:
- To identify specific residues involved in the FusB-EF-G interaction.
- To delineate the binding interface between FusB and EF-G.
- To understand the molecular basis of fusidic acid resistance.
Main Methods:
- Site-directed mutagenesis of conserved residues in FusB (F156, K184, Y187, F208) to alanine.
- In vitro binding assays using purified wild-type and mutant FusB proteins with Staphylococcus aureus EF-G.
- Genetic modification of Escherichia coli EF-G by deleting residues 529-531 to assess binding competency with FusB.
Main Results:
- Mutating four conserved FusB residues (F156, K184, Y187, F208) abolished fusidic acid resistance and in vitro binding to S. aureus EF-G.
- Deletion of residues 529-531 in E. coli EF-G domain IV enabled binding to FusB.
- These findings pinpoint critical interaction sites on both FusB and EF-G.
Conclusions:
- Specific residues in FusB and EF-G are essential for their interaction.
- The identified binding interface provides insight into the mechanism of fusidic acid resistance mediated by FusB proteins.
- This work validates previous in silico predictions regarding the FusB-EF-G complex structure.
