Structural insights into fusidic acid resistance and sensitivity in EF-G.
Fusidic acid (FA) is a steroid antibiotic commonly used against Gram positive bacterial infections. It inhibits protein synthesis by stalling elongation factor G (EF-G) on the ribosome after translocation. A significant number of the mutations conferring strong FA resistance have been mapped at the interfaces between domains G, III and V of EF-G. However, direct information on how such mutations affect the structure has hitherto not been available. Here we present the crystal structures of two mutants of Thermus thermophilus EF-G, G16V and T84A, which exhibit FA hypersensitivity and resistance in vitro, respectively. These mutants also have higher and lower affinity for GTP respectively than wild-type EF-G. The mutations cause significant conformational changes in the switch II loop that have opposite effects on the position of a key residue, Phe90, which undergoes large conformational changes. This correlates with the importance of Phe90 in FA sensitivity reported in previous studies. These structures substantiate the importance of the domain G/domain III/domain V interfaces as a key component of the FA binding site. The mutations also cause subtle changes in the environment of the "P-loop lysine", Lys25. This led us to examine the conformation of the equivalent residue in all structures of translational GTPases, which revealed that EF-G and eEF2 form a group separate from the others and suggested that the role of Lys25 may be different in the two groups.
Hansson
Sebastian
S
Department of Molecular Biophysics, Lund University, Box 124, S-221 00 Lund, Sweden.
Singh
Ranvir
R
Gudkov
Anatoly T
AT
Liljas
Anders
A
Logan
Derek T
DT
D018360:Crystallography, X-Ray; D024881:Drug Resistance, Bacterial / Q000187:drug effects*; D005672:Fusidic Acid / Q000737:chemistry / Q000494:pharmacology*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D020653:Peptide Elongation Factor G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000737:chemistry* / Q000235:genetics
D020653:Peptide Elongation Factor G; D006153:Guanosine Diphosphate; D005672:Fusidic Acid; D006160:Guanosine Triphosphate; D008239:Lysine