title: GE2270A-resistant mutations in elongation factor Tu allow productive aminoacyl-tRNA binding to EF-Tu.GTP.GE2270A complexes.
abstract: 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.
authors: Department of Biochemistry Leiden Institute of Chemistry, Leiden University, Leiden, 2300 RA, The Netherlands.
authors: A M
authors: AM
authors: Zuurmond
authors: J
authors: J
authors: Martien de Graaf
authors: L N
authors: LN
authors: Olsthoorn-Tieleman
authors: B Y
authors: BY
authors: van Duyl
authors: V G
authors: VG
authors: Mörhle
authors: F
authors: F
authors: Jurnak
authors: J R
authors: JR
authors: Mesters
authors: R
authors: R
authors: Hilgenfeld
authors: B
authors: B
authors: Kraal
mesh_terms: D000192:Actinomycetales / Q000737:chemistry; D000225:Adenine / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010456:Peptides, Cyclic / Q000737:chemistry / Q000494:pharmacology*; D011072:Poly U / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D013824:Thermus / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D010455:Peptides; D010456:Peptides, Cyclic; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D013844:Thiazoles; C012521:pulvomycin; D006153:Guanosine Diphosphate; D011072:Poly U; C012001:polyphenylalanine; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu; D000225:Adenine; C068122:GE 2270 A