RESUMO
The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E. coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3' end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu.GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu.GDP.GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu.GTP.GE2270.aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.
Assuntos
Aminoglicosídeos , Escherichia coli , Guanosina Trifosfato/metabolismo , Mutação/genética , Fator Tu de Elongação de Peptídeos/genética , Fator Tu de Elongação de Peptídeos/metabolismo , Peptídeos Cíclicos/farmacologia , Aminoacil-RNA de Transferência/metabolismo , Tiazóis/metabolismo , Actinomycetales/química , Adenina/metabolismo , Substituição de Aminoácidos/genética , Antibacterianos/química , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Guanosina Difosfato/metabolismo , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/química , Peptídeos/metabolismo , Peptídeos Cíclicos/química , Poli U/genética , Poli U/metabolismo , Ligação Proteica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Conformação Proteica , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Aminoacil-RNA de Transferência/genética , Termodinâmica , Thermus/química , Tiazóis/química , Tiazóis/farmacologiaRESUMO
For clarification of the action of a new antibiotic, the analysis of resistant mutants is often indispensable. For enacyloxin IIa we discovered four resistant elongation factor Tu (EF-Tu) species in Escherichia coli with the mutations Q124K, G316D, Q329H, and A375T, respectively. They revealed that enacyloxin IIa sensitivity is dominant in a mixed population of resistant and wild-type EF-Tus. This points to an inhibition mechanism in which EF-Tu is the dominant target of enacyloxin IIa and in which a ribosome with a sensitive EF-Tu blocks mRNA translation for upstream ribosomes with resistant EF-Tus, a mechanism similar to that of the unrelated antibiotic kirromycin. Remarkably, the same mutations are also linked to kirromycin resistance, though the order of their levels of resistance is different from that for enacyloxin IIa. Among the mutant EF-Tus, three different resistance mechanisms can be distinguished: (i) by obstructing enacyloxin IIa binding to EF-Tu. GTP; (ii) by enabling the release of enacyloxin IIa after GTP hydrolysis; and (iii) by reducing the affinity of EF-Tu.GDP. enacyloxin IIa for aminoacyl-tRNA at the ribosomal A-site, which then allows the release of EF-Tu.GDP.enacyloxin IIa. Ala375 seems to contribute directly to enacyloxin IIa binding at the domain 1-3 interface of EF-Tu.GTP, a location that would easily explain the pleiotropic effects of enacyloxin IIa on the functioning of EF-Tu.
Assuntos
Fator Tu de Elongação de Peptídeos/genética , Ribossomos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Modelos Moleculares , Fator Tu de Elongação de Peptídeos/metabolismo , Fenilalanina/biossíntese , Polienos/metabolismo , Polímeros/metabolismo , Biossíntese de Proteínas , Piridonas/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Relação Estrutura-AtividadeRESUMO
Fluoraluminates are thought to mimic the gamma-phosphate of GTP and thus, together with GDP, perturb the functioning of heterotrimeric GTP-binding G-proteins. Here we show they do inhibit the ribosome-stimulated GTPase activity of EF-G from Escherichia coli via the formation of a stable complex with EF-G-GDP and ribosomes. In contrast, no perturbed interactions were observed in a similar ribosomal complex with EF-Tu. Interestingly, in the absence of ribosomes both EF-Tu an EF-G remain totally unaffected by fluoraluminates. For members of the GTPase superfamily such differential effects have not been described before.