Crystal structure and site-directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering.
The antibiotic bleomycin, a strong DNA cutting agent, is naturally produced by actinomycetes which have developed a resistance mechanism against such a lethal compound. The crystal structure, at 2.3 A resolution, of a bleomycin resistance protein of 14 kDa reveals a structure in two halves with the same alpha/beta fold despite no sequence similarity. The crystal packing shows compact dimers with a hydrophobic interface and involved in mutual chain exchange. Two independent solution studies (analytical centrifugation and light scattering) showed that this dimeric form is not a packing artefact but is indeed the functional one. Furthermore, light scattering also showed that one dimer binds two antibiotic molecules as expected. A crevice located at the dimer interface, as well as the results of a site-directed mutagenesis study, led to a model wherein two bleomycin molecules are completely sequestered by one dimer. This provides a novel insight into antibiotic resistance due to drug sequestering, and probably also into drug transport and excretion.
Dumas
P
P
Laboratoire de Biologie Structurale, CNRS, Strasbourg, France.
Bergdoll
M
M
Cagnon
C
C
Masson
J M
JM
D000123:Acetyltransferases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001761:Bleomycin / Q000378:metabolism* / Q000494:pharmacology; D003460:Crystallization; D018360:Crystallography, X-Ray; D004352:Drug Resistance, Microbial / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D001761:Bleomycin; C087954:bleomycin N-acetyltransferase; D000123:Acetyltransferases