Crystal Structures of the Transposon Tn5-carried Bleomycin Resistance Determinant Uncomplexed and Complexed with Bleomycin

Abstract

The transposon Tn5 carries a gene designated ble that confers resistance to bleomycin (Bm). In this study, we determined the x-ray crystal structures of the ble gene product, designated BLMT, uncomplexed and complexed with Bm at 1.7 and 2.5 Å resolution, respectively. The structure of BLMT is a dimer with two Bm-binding pockets composed of two large concavities and two long grooves. This crystal structure of BLMT complexed with Bm gives a precise mode for binding of the antibiotic to BLMT. The conformational change of BLMT generated by binding to Bm occurs at a ␤-turn composed of the residues from Gln 97 to Thr 102 . Crystallographic analysis of Bm bound to BLMT shows that two thiazolium rings of the bithiazole moiety are in the trans conformation. The axial ligand, which binds a metal ion, seems to be the primary amine in the ␤-aminoalanine moiety. This report, which is the first with regard to the x-ray crystal structure of Bm, shows that the bithiazole moiety of Bm is far from the metal-binding domain. That is, Bm complexed with BLMT takes a more extended form than the drug complexed with DNA.

Characterization of BRP MBL , the Bleomycin Resistance Protein Associated with the Carbapenemase NDM

“…Those proteins, even though sharing, respectively, 18%, 52%, and 16% amino acid identity with BRP MBL only, have similar three-dimensional (3D) structures, suggesting that they may be used as bases for BRP MBL modeling. The crystal structures are dimers, and a few of them are complexed with two bleomycin molecules, which are located symmetrically at the monomermonomer interfaces (13). In silico modeling suggested that the three-dimensional model of the BRP MBL needed to be constructed directly as a dimer, in the presence of the bleomycin ligand.…”

“…The three-dimensional model of BRP MBL was built using Modeler v9.16 (28), with the structure of BMLT (PDB code 1EWJ) as the template (13). The structure was constructed as a dimer, with two bleomycin molecules positioned at the interface of the monomers.…”

“…The capacity of binding of the GST-BRP MBL to bleomycin was assayed by electrophoretic mobility shift assay (EMSA) by using purified GST-BRP MBL (20 g) and 20 g bleomycin or zeocin in 30 l of 10 mM Tris-HCl (pH 7.5) at 16°C. After 3 h of incubation, 30 l of the reaction mixture was loaded onto a 10% nondenaturing polyacrylamide gel, as previously described (13,23,24).…”

Transcription-Dependent Increase in Multiple Classes of Base Substitution Mutations in Escherichia coli

“…The likely reason that the original amino acid at position 75, Gln, can be replaced with any of six other amino acids is that this residue is not critically involved in the binding of the antibiotic (15).…”

The 1.6-Å Crystal Structure of the Copper(II)-bound Bleomycin Complexed with the Bleomycin-binding Protein from Bleomycin-producing Streptomyces verticillus

“…However, the x-ray crystallographic analysis of Bm had not been successful until our group (9) determined the x-ray crystal structure of Bm complexed with the transposon Tn5-carried Bm-binding protein.…”