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PMID: 7525272 Published · ppublish English Journal Article

The structural and functional basis for the kirromycin resistance of mutant EF-Tu species in Escherichia coli.

The EMBO journal ·Vol. 13 ·No. 20 ·1994-10-17 ·Pages 4877-85

Mesters JR, Zeef LA, Hilgenfeld R, de Graaf JM, Kraal B, Bosch L

Abstract

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.

MeSH Terms
Anti-Bacterial Agents/metabolism,pharmacology Drug Resistance, Microbial/genetics Escherichia coli/drug effects,genetics Guanosine Triphosphate/metabolism Models, Molecular Mutation Peptide Elongation Factor Tu/genetics,metabolism,physiology Protein Binding Pyridones/metabolism,pharmacology RNA, Bacterial/metabolism RNA, Transfer, Amino Acyl/metabolism Ribosomes/metabolism Structure-Activity Relationship
Chemicals
Anti-Bacterial Agents Pyridones RNA, Bacterial RNA, Transfer, Amino Acyl Guanosine Triphosphate Peptide Elongation Factor Tu mocimycin
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Mesters J R
Department of Biochemistry, Leiden University, The Netherlands.
Zeef L A
Hilgenfeld R
de Graaf J M
Kraal B
Bosch L
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1994-10-17
Pages
4877-85
Language
English
Region
England
NLM ID
8208664
PMCID
PMC395428
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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