The structural and functional basis for the kirromycin resistance of mutant EF-Tu species in Escherichia coli.
A structural and functional understanding of resistance to the antibiotic kirromycin in Escherichia coli has been sought in order to shed new light on the functioning of the bacterial elongation factor Tu (EF-Tu), in particular its ability to act as a molecular switch. The mutant EF-Tu species G316D, A375T, A375V and Q124K, isolated by M13mp phage-mediated targeted mutagenesis, were studied. In this order the mutant EF-Tu species showed increasing resistance to the antibiotic as measured by poly(U)-directed poly(Phe) synthesis and intrinsic GTPase activities. The K'd values for kirromycin binding to mutant EF-Tu.GTP and EF-Tu.GDP increased in the same order. All mutation sites cluster in the interface of domains 1 and 3 of EF-Tu.GTP, not in that of EF-Tu.GDP. Evidence is presented that kirromycin binds to this interface of wild-type EF-Tu.GTP, thereby jamming the conformational switch of EF-Tu upon GTP hydrolysis. We conclude that the mutations result in two separate mechanisms of resistance to kirromycin. The first inhibits access of the antibiotic to its binding site on EF-Tu.GTP. A second mechanism exists on the ribosome, when mutant EF-Tu species release kirromycin and polypeptide chain elongation continues.
Mesters
J R
JR
Department of Biochemistry, Leiden University, The Netherlands.
Zeef
L A
LA
Hilgenfeld
R
R
de Graaf
J M
JM
Kraal
B
B
Bosch
L
L
D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D011728:Pyridones / Q000378:metabolism / Q000494:pharmacology; D012329:RNA, Bacterial / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D011728:Pyridones; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; C008536:mocimycin