The mitomycin C (MMC)-binding protein from MMC-producing microorganisms protects from the lethal effect of bleomycin: crystallographic analysis to elucidate the binding mode of the antibiotic to the protein

Antibiotic-producing microorganisms must be protected from the lethal effect of their own antibiotic. We have previously determined the X-ray crystal structure of the bleomycin (Bm)-binding protein, designated BLMA, as a self-resistance determinant from Bm-producing Streptomyces verticillus, which suggests that the binding of the first Bm to one of two pockets formed in the BLMA homodimer induces the cooperative binding of the second Bm to the other pocket. In the present study, we noticed that the X-ray crystallographic structure of a self-resistance determinant from a mitomycin C-producing microorganism, designated MRDP, reveals similarity to the folding pattern on the BLMA, although no sequence homology exists. To clarify the hypothesis that MRDP may function as a resistance determinant to Bm, we characterized and determined the crystal structure of MRDP complexed with the Cu(II)-bound form of BmA(2) grouped into the Bm family of antibiotics. The biochemical and structural studies for Bm binding provide evidence that the first Bm binds anti-cooperatively to a pocket of MRDP with binding affinity of the nanomolar order, whereas the second Bm binds to the other pocket, which has binding affinity of the micromolar order. The invisibility of the second Bm in the structure agrees with the observation that Escherichia coli-expressing MRDP displays lower resistance to Bm than that expressing BLMA. The structure of MRDP, which is complexed with the Cu(II)-bound BmA(2), revealed that the gamma-aminopropyldimethylsulphonium moiety of the antibiotic is sandwiched between the peripheral residues of the binding pocket and that its positively charged sulphonium head is accommodated completely in the negatively charged region of the MRDP pocket. Furthermore, the Cu(II)-bound BmA(2) has a very compact structure, in which the bithiazole ring of BmA(2) is folded back to the metal-binding domain.     

The 1.6-A crystal structure of the copper(II)-bound bleomycin complexed with the bleomycin-binding protein from bleomycin-producing Streptomyces verticillus.

Bleomycin (Bm) in the culture broth of Streptomyces verticillus is complexed with Cu(2+) (Cu(II)). In the present study, we determined the x-ray crystal structures of the Cu(II)-bound and the metal-free types of Bm at a high resolution of 1.6 and 1.8 A, respectively, which are complexed with a Bm resistance determinant from Bm-producing S. verticillus, designated BLMA. In the current model of Cu(II).Bm complexed with BLMA, two Cu(II).Bm molecules bind to the BLMA dimer. The electron density map shows that the copper ion is clearly defined in the metal-binding domain of the Bm molecule. The metal ion is penta-coordinated by a tetragonal monopyramidal cage of nitrogens and binds to the primary amine of the beta-aminoalanine moiety of Bm. The binding experiment between Bm and BLMA showed that each of the two Bm-binding pockets has a different dissociation constant (K(d)(1) and K(d)(2)). The K(d)(1) value of 630 nm for the first Bm binding is larger than the K(d)(2) value of 120 nm, indicating that the first Bm binding gives rise to a cooperative binding of the second Bm to the other pocket.     

NMR study of the interaction between Zn(II) ligated bleomycin and Streptoalloteichus hindustanus bleomycin resistance protein

Bleomycin (Bm), a 1.4 kDa glycopeptide excreted by Streptomyces verticillus, is a natural antibacterial compound used in therapy as antineoplastic drug. To counteract its biological activity, cells have developed several resistance mechanisms, one of these based on proteins able to tightly bind Bm. In this paper, the interaction of Zn(2+)-Bm with the Streptoalloteichus hindustanus Bm resistance protein (ShBle) has been investigated by solution state NMR. Sequential nOe and chemical shift index have shown that the fold of the protein (in absence or presence of Bm) is identical to the previously published X-ray structure. The dimeric nature of ShBle is confirmed by the diffusion tensor as determined by NMR relaxation data. Using isotope filtered nOe experiment, intermolecular nOes between Bm and ShBle have been observed as used for modeling. While the interaction of the Bm metal binding site with ShBle appears to be uniquely defined, several conformations of the bithiazole moieties are compatible with the NMR data. Binding of Bm also induces changes of the local dynamics (stretch N85-G91), as shown by (15)N relaxation data. These results are discussed in the context of several Bm analogues able to interact with ShBle and of the recently published X-rays structures.     

Optimized vectors and selection for transformation of Neurospora crassa and Aspergillus nidulans to bleomycin and phleomycin resistance.

To provide a dominant selectable marker for transformation of Neurospora crassa strains lacking specific auxotrophic mutations, we have engineered the bleomycin (Bm) resistance-encoding gene (ble) from the bacterial transposon Tn5 for expression in N. crassa. The coding region of the ble gene was fused to the promoter and terminator regions of the N. crassa am gene. In some vectors, multiple cloning sites were placed flanking the ble gene to provide a versatile ble cassette. When introduced into N. crassa, the hybrid ble gene conferred resistance to greater than 15 micrograms Bm/ml. Under optimal conditions, the levels of Bm required (2.5 micrograms/ml) make even large-scale transformation experiments very economical. Aspergillus nidulans could also be efficiently transformed to Bm resistance using the N. crassa ble gene fusion. Since the ble gene functions in both N. crassa and A. nidulans, the gene should be useful as a transformation marker for the many other filamentous fungi which are sensitive to Bm.     

The presence of two modes of binding to calf thymus DNA by metal-free bleomycin: a low frequency Raman study

Double-stranded DNA is targeted by bleomycin in cancer cells and ambiguity exists as to its mode of DNA binding. A conventional Raman study was performed on drug/DNA complexes in which the low frequency spectral region (560-930 cm(-1)) was examined at two temperatures (19 and 30 degrees C). At 30 degrees C, a global Raman hypochromism was observed consistent with partial intercalation of the bithiazole moiety. At 19 degrees C, Raman hypochromism (increased base pair stacking) was detected for bands associated with GC base pairs while Raman hyperchromism (base pair destacking) was evident for bands associated with AT base pairs. These results suggest that intercalation of the bithiazole moiety occurs with greater disruption of the more efficiently stacked AT base pairs at the lower temperature. Evidence for minor groove binding was indicated by an increase in the population of bands corresponding to C3' endo sugar conformations resulting from drug induced local desolvation of the DNA polymer.     

The 1.5 A crystal structure of a bleomycin resistance determinant from bleomycin-producing Streptomyces verticillus

Bleomycin (Bm)-binding protein, designated BLMA, which is a Bm resistance determinant from Bm-producing Streptomyces verticillus, was crystallized in a form suitable for X-ray diffraction analysis. The diffraction intensity data were collected up to a resolution of 1.5 A with a merging R-value of 0.054 at a completeness of 94 %. The BLMA structure, determined by the single isomorphous replacement method including the anomalous scattering effect (SIR-AS) at a resolution of 2.0 A, was refined at 1.5 A resolution. The final R-factor was 19.0 % and R(free) was 22.1 % including 91 water molecules. The crystal packing showed a dimer form, which was generated by arm exchange. The 1.5 A high-resolution experiment allowed an analysis of the side-chain disorder of BLMA. The structural comparison of BLMA with a homologous protein from Streptoalloteichus hindustanus, designated Shble protein, showed that a Ser100-Gly103 loop was farther from the groove, which is a Bm-binding site, in BLMA than in the Shble protein. Furthermore the hydrophobicity of the groove in BLMA is much lower than that in the Shble protein. The structural differences between these proteins may be responsible for the observation that a half-saturating concentration (K(1/2)) of Bm is higher for BLMA than for the Shble protein.     

Binding properties and DNA sequence-specific recognition of two bithiazole-linked netropsin hybrid molecules.

We report the DNA binding properties of two hybrid molecules which result from the combination of the DNA sequence-specific minor groove ligand netropsin with the bithiazole moiety of the antitumor drug bleomycin. The drug-DNA interaction has been investigated by means of electric linear dichroism (ELD) spectroscopy and DNase I footprinting. In compound 1 the two moieties are linked by a flexible aliphatic tether while in compound 2 the two aromatic ring systems are directly coupled by a rigid peptide bond. The results are consistent with a model in which the netropsin moiety of compound 1 resides in the minor groove of DNA and where the appended bithiazole moiety is projected away from the DNA groove. This monocationic hybrid compound has a weak affinity for DNA and shows a strict preference for A and T stretches. ELD measurements indicate that in the presence of DNA compound 2 has an orientation typical of a minor groove binder. Similar orientation angles were measured for netropsin and compound 2. This ligand which has a biscationic nature tightly binds to DNA (Ka = 6.3 x 10(5) M-1) and is mainly an AT-specific groove binder. But, depending on the nature of the sequence flanking the AT site first targeted by its netropsin moiety, the bithiazole moiety of 2 can accommodate various types of nucleotide motifs with the exception of homooligomeric sequences. As evidenced by footprinting data, the bithiazole group of bleomycin acts as a DNA recognition element, offering opportunities to recognize GC bp-containing DNA sequences with apparently a preference (although not absolute) for a pyrimidine-G-pyrimidine motif. Thus, the bithiazole unit of bleomycin provides an additional anchor for DNA binding and is also capable of specifically recognizing particular DNA sequences when it is appended to a strongly sequence selective groove binding entity. Finally, a model which schematizes the binding of compound 2 to the sequence 5'-TATGC is proposed. This model readily explains the experimentally observed specificity of this netropsin-bithiazole conjugate.     

Models of bleomycin interactions with poly(deoxyadenylylthymidylic acid). Fluorescence and proton nuclear magnetic resonance studies of cationic thiazole amides related to bleomycin A2

The interaction of eight 2-substituted thiazole-4-carboxamides, structurally related to cationic terminus of bleomycin A2, with poly(deoxyadenylylthymidylic acid) [poly(dA-dT)] has been studied by using proton nuclear magnetic resonance and fluorescence spectroscopy. These analogues have been used as probes of the complex formed between the parent drug molecule and poly(dA-dT). Aliphatic substituents on the 2' position of 2,4'-bithiazole derivatives restrict the ability of the aromatic ring system to intercalate in the double-helical form of the polynucleotide. Absence or partial removal of the 2' substituent enhances intercalation of the bithiazole system. The cationic side chain does not appear to be involved in the stabilization of any of these complexes, although it may be necessary for their formation. A 2,4':2',4"-terthiazole derivative shows a substantial degree of intercalation which is accompanied by extensive immobilization of the cationic side chain. This suggests that insertion of the aromatic system into the nucleic acid causes the cationic side chain to be pulled in also. Monothiazole analogues do not appear to bind, indicating that at least two thiazole rings are necessary for binding or that proper spacing between the two side chains on either side of the thiazole system is important for binding. The relation of the interactions of these analogues to the biochemical and biological properties of the parent bleomycins is discussed as is the possible use of these data in the design of synthetic bleomycin derivatives having varying affinities and specificities for DNA.     

Novel DNA photocleaving agents with high DNA sequence specificity related to the antibiotic bleomycin A2.

We have designed and synthesized a series of novel DNA photocleaving agents which break DNA with high sequence specificity. These compounds contain the non-diffusible photoactive p-nitrobenzoyl group covalently linked via a dimethylene (or tetramethylene) spacer to thiazole analogues of the DNA binding portion of the antibiotic bleomycin A2. By using a variety of 5' or 3' 32P-end labeled restriction fragments from plasmid pBR322 as substrate, we have shown that photoactive bithiazole compounds bind DNA at the consensus sequence 5'-AAAT-3' and induce DNA cleavage 3' of the site. Analysis of cleavage sites on the complementary DNA strand and inhibition of DNA breakage by distamycin A indicates these bithiazole derivatives bind and attack the minor groove of DNA. A photoactive unithiazole compound was less specific inducing DNA breakage at the degenerate site 5'-(A/T)(AA/TT)TPu(A/T)-3'. DNA sequence recognition of these derivatives appears to be determined by the thiazole moiety rather than the p-nitrobenzoyl group: use of a tetramethylene group in place of a dimethylene spacer shifted the position of DNA breakage by one base pair. Moreover, much less specific DNA photocleavage was observed for a compound in which p-nitrobenzoyl was linked to the intercalator acridine via a sequence-neutral hexamethylene spacer. The 5'-AAAT-3' specificity of photoactive bithiazole derivatives contrasts with that of bleomycin A2 which cleaves DNA most frequently at 5'-GPy-3' sequences. These results suggest that the cleavage specificity exhibited by bleomycin is not simply determined by its bithiazole/sulphonium terminus, and the contributions from other features, e.g. its metal-chelating domain, must be considered. The novel thiazole-based DNA cleavage agents described here should prove useful as reagents for probing DNA structure and for elucidating the molecular basis of DNA recognition by bleomycin and other ligands.     

Mutation of the N-terminal proline 9 of BLMA from Streptomyces verticillus abolishes the binding affinity for bleomycin

A gene, blmA, from bleomycin (Bm)-producing Streptomyces verticillus, encodes a Bm-binding protein, designated BLMA. The expression of BLMA conferred resistance to Bm in the Escherichia coli host, whereas a mutant protein, designated Pro-9/Leu, with the N-terminal proline 9 residue in BLMA replaced by leucine, did not. We created a fusion protein between the maltose-binding protein (MBP) and a mutant protein Pro-9/Leu/Leu with Met-94 in Pro-9/Leu replaced by leucine. Pro-9/Leu/Leu from the fusion protein, obtained by digestion with CNBr digestion, did not inhibit DNA-cleaving and antibacterial activities of Bm. Native-polyacrylamide gel electrophoresis (PAGE) and gel filtration column chromatographic analysis showed that the molecular size of Pro-9/Leu/Leu is roughly half of that of BLMA, suggesting that the mutant protein cannot form dimeric structure. Furthermore, Far-UV circular dichroism (CD) spectrum of Pro-9/Leu/Leu was quite different from that of BLMA and similar to the spectra obtained from unordered proteins [Venyaminov, S.Y. and Vassilenko, K.S. (1994) Anal. Biochem. 222, 176–184], suggesting that the secondary structure of Pro-9/Leu/Leu is disrupted. These results indicate that the mutation abolishes not only dimer formation but also the secondary structure of BLMA, which results in the loss of its function as a Bm-resistance determinant.     

Mutation of the N-terminal proline 9 of BLMA from Streptomyces verticillus abolishes the binding affinity for bleomycin.

A gene, blmA, from bleomycin (Bm)-producing Streptomyces verticillus, encodes a Bm-binding protein, designated BLMA. The expression of BLMA conferred resistance to Bm in the Escherichia coli host, whereas a mutant protein, designated Pro-9/Leu, with the N-terminal proline 9 residue in BLMA replaced by leucine, did not. We created a fusion protein between the maltose-binding protein (MBP) and a mutant protein Pro-9/Leu/Leu with Met-94 in Pro-9/Leu replaced by leucine. Pro-9/Leu/Leu from the fusion protein, obtained by digestion with CNBr digestion, did not inhibit DNA-cleaving and antibacterial activities of Bm. Native-polyacrylamide gel electrophoresis (PAGE) and gel filtration column chromatographic analysis showed that the molecular size of Pro-9/Leu/Leu is roughly half of that of BLMA, suggesting that the mutant protein cannot form dimeric structure. Furthermore, Far-UV circular dichroism (CD) spectrum of Pro-9/Leu/Leu was quite different from that of BLMA and similar to the spectra obtained from unordered proteins [Venyaminov, S.Y. and Vassilenko, K.S. (1994) Anal. Biochem. 222, 176184], suggesting that the secondary structure of Pro-9/Leu/Leu is disrupted. These results indicate that the mutation abolishes not only dimer formation but also the secondary structure of BLMA, which results in the loss of its function as a Bm-resistance determinant.     

Apo- and holo-structures of 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831. Loop-helix transition induced by coenzyme binding

Bacterial 3alpha-hydroxysteroid dehydrogenase, which belongs to a short-chain dehydrogenase/reductase family and forms a dimer composed of two 26-kDa subunits, catalyzes the oxidoreduction of hydroxysteroids in a coenzyme-dependent manner. This enzyme also catalyzes the oxidoreduction of nonsteroid compounds that play an important role in xenobiotic metabolism of bacteria. We performed an x-ray analysis on the crystal of Ps3alphaHSD, the enzyme from Pseudomonas sp. B-0831 complexed with NADH. The resulting crystal structure at 1.8A resolution showed that Ps3alphaHSD exists as a structural heterodimer composed of apo- and holo-subunits. A distinct structural difference between them was found in the 185-207-amino acid region, where the structure in the apo-subunit is disordered whereas that in the holo-subunit consists of two alpha-helices. This fact proved that the NADH binding allows the helical structures to form the substrate binding pocket even in the absence of the substrate, although the region corresponds to the so-called "substrate-binding loop." The induction of alpha-helices in solution by the coenzyme binding was also confirmed by the CD experiment. In addition, the CD experiment revealed that the helix-inducing ability of NADH is stronger than that of NAD. We discuss the negative cooperativity for the coenzyme binding, which is caused by the effect of the structural change transferred between the subunits of the heterodimer.     

Photoactivated DNA cleavage by compounds structurally related to the bithiazole moiety of bleomycin.

The syntheses of several novel halogenated bithiazoles structurally related to the bithiazole moiety of bleomycin A(5) are described. Also described is the ability of these compounds to mediate photoactivated DNA cleavage. Chlorinated bithiazole analogues were shown to be much more active than an analogous brominated derivative. DNA strand scission activity was strictly light dependent and was accompanied by dechlorination of the bithiazole nucleus, apparently in a stoichiometric fashion. Inhibition of DNA cleavage in the presence of DMSO, as well as photoaddition to 1-octene by both brominated and chlorinated bithiazole derivatives, suggest strongly that the initial step in photoactivated DNA cleavage involves homolysis of the thiazole carbon-halogen bond. The chlorinated bithiazoles were found to mediate sequence selective cleavage of a (32)P-end labeled DNA, although the selectivity observed was not the same as that of bleomycin itself. The implications of this observation are discussed.     

SAR and factor IXa crystal structure of a dual inhibitor of factors IXa and Xa

Modifications to the P4 moiety and pyrazole C3 substituent of factor Xa inhibitor SN-429 provided several new compounds, which are 5-10nM inhibitors of factor IXa. An X-ray crystal structure of one example complexed to factor IXa shows that these compounds adopt a similar binding mode to that previously observed with pyrazole inhibitors in the factor Xa active site both with regard to how the inhibitor binds and the position of Tyr99.     

Interaction of bleomycin A2 with deoxyribonucleic acid: DNA unwinding and inhibition of bleomycin-induced DNA breakage by cationic thiazole amides related to bleomycin A2

The association of the antitumor antibiotic bleomycin A2 with DNA has been investigated by employing several 2-substituted thiazole-4-carboxamides, structurally related to the cationic terminus of the drug. With a 5'-32P-labeled DNA restriction fragment from plasmid pBR322 as substrate, these compounds have been shown to inhibit bleomycin-induced DNA breakage. Analogues possessing 2'-aromatic substituents on the bithiazole ring were more potent inhibitors than those carrying 2'-aliphatic groups, e.g., the acetyl dipeptide A2. The degree of inhibition was similar at all scission sites on DNA, and inclusion of the analogues did not induce bleomycin cleavage at new sites. DNA binding of bithiazole derivatives has also been studied by two complementary topological methods. Two-dimensional gel electrophoresis using a population of DNA topoisomers and DNA relaxation experiments involving calf thymus DNA topoisomerase I and pBR322 DNA reveal that bleomycin bithiazole analogues unwind closed circular duplex DNA. The inhibition and unwinding studies together support recent NMR studies suggesting that both bleomycin A2 and synthetic bithiazole derivatives bind to DNA by an intercalative mechanism. The results are discussed in relation to the DNA breakage properties of bleomycin A2.     

Intermediate states of ribonuclease III in complex with double-stranded RNA

Bacterial ribonuclease III (RNase III) can affect RNA structure and gene expression in either of two ways: as a processing enzyme that cleaves double-stranded (ds) RNA, or as a binding protein that binds but does not cleave dsRNA. We previously proposed a model of the catalytic complex of RNase III with dsRNA based on three crystal structures, including the endonuclease domain of RNase III with and without bound metal ions and a dsRNA binding protein complexed with dsRNA. We also reported a noncatalytic assembly observed in the crystal structure of an RNase III mutant, which binds but does not cleave dsRNA, complexed with dsRNA. We hypothesize that the RNase III*dsRNA complex can exist in two functional forms, a catalytic complex and a noncatalytic assembly, and that in between the two forms there may be intermediate states. Here, we present four crystal structures of RNase III complexed with dsRNA, representing possible intermediates.     

A novel adenylate binding site confers phosphopantetheine adenylyltransferase interactions with coenzyme A

Phosphopantetheine adenylyltransferase (PPAT) regulates the key penultimate step in the essential coenzyme A (CoA) biosynthetic pathway. PPAT catalyzes the reversible transfer of an adenylyl group from Mg(2+):ATP to 4'-phosphopantetheine to form 3'-dephospho-CoA (dPCoA) and pyrophosphate. The high-resolution crystal structure of PPAT complexed with CoA has been determined. Remarkably, CoA and the product dPCoA bind to the active site in distinct ways. Although the phosphate moiety within the phosphopantetheine arm overlaps, the pantetheine arm binds to the same pocket in two distinct conformations, and the adenylyl moieties of these two ligands have distinct binding sites. Moreover, the PPAT:CoA crystal structure confirms the asymmetry of binding to the two trimers within the hexameric enzyme. Specifically, the pantetheine arm of CoA bound to one protomer within the asymmetric unit displays the dPCoA-like conformation with the adenylyl moiety disordered, whereas CoA binds the twofold-related protomer in an ordered and unique fashion.     

Crystal structure of perakine reductase, founding member of a novel aldo-keto reductase (AKR) subfamily that undergoes unique conformational changes during NADPH binding

Perakine reductase (PR) catalyzes the NADPH-dependent reduction of the aldehyde perakine to yield the alcohol raucaffrinoline in the biosynthetic pathway of ajmaline in Rauvolfia, a key step in indole alkaloid biosynthesis. Sequence alignment shows that PR is the founder of the new AKR13D subfamily and is designated AKR13D1. The x-ray structure of methylated His(6)-PR was solved to 2.31 ?. However, the active site of PR was blocked by the connected parts of the neighbor symmetric molecule in the crystal. To break the interactions and obtain the enzyme-ligand complexes, the A213W mutant was generated. The atomic structure of His(6)-PR-A213W complex with NADPH was determined at 1.77 ?. Overall, PR folds in an unusual α(8)/β(6) barrel that has not been observed in any other AKR protein to date. NADPH binds in an extended pocket, but the nicotinamide riboside moiety is disordered. Upon NADPH binding, dramatic conformational changes and movements were observed: two additional β-strands in the C terminus become ordered to form one α-helix, and a movement of up to 24 ? occurs. This conformational change creates a large space that allows the binding of substrates of variable size for PR and enhances the enzyme activity; as a result cooperative kinetics are observed as NADPH is varied. As the founding member of the new AKR13D subfamily, PR also provides a structural template and model of cofactor binding for the AKR13 family.