Mutations in the G-domain of elongation factor G from Thermus thermophilus affect both its interaction with GTP and fusidic acid.
Two hypersensitive and two resistant variants of elongation factor-G (EF-G) toward fusidic acid are studied in comparison with the wild type factor. All mutated proteins are active in a cell-free translation system and ribosome-dependent GTP hydrolysis. The EF-G variants with the Thr-84-->Ala or Asp-109-->Lys mutations bring about a strong resistance of EF-G to the antibiotic, whereas the EF-Gs with substitutions Gly-16-->Val or Glu-119-->Lys are the first examples of fusidic acid-hypersensitive factors. A correlation between fusidic acid resistance of EF-G mutants and their affinity to GTP are revealed in this study, although their interactions with GDP are not changed. Thus, fusidic acid-hypersensitive mutants have the high affinity to an uncleavable GTP analog, but the association of resistant mutants with GTP is decreased. The effects of either fusidic acid-sensitive or resistant mutations can be explained by the conformational changes in the EF-G molecule, which influence its GTP-binding center. The results presented in this paper indicate that fusidic acid-sensitive mutant factors have a conformation favorable for GTP binding and subsequent interaction with the ribosomes.
Martemyanov
K A
KA
Institute of Protein Research, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia.
Liljas
A
A
Yarunin
A S
AS
Gudkov
A T
AT
D000409:Alanine; D001224:Aspartic Acid; D001665:Binding Sites; D002474:Cell-Free System; D005672:Fusidic Acid / Q000378:metabolism*; D014644:Genetic Variation; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D008239:Lysine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020653:Peptide Elongation Factor G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011072:Poly U / Q000378:metabolism; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*; D013912:Threonine
D020653:Peptide Elongation Factor G; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D011072:Poly U; D013912:Threonine; D001224:Aspartic Acid; D006165:Guanylyl Imidodiphosphate; D005672:Fusidic Acid; D006160:Guanosine Triphosphate; D008239:Lysine; D000409:Alanine