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

Masafumi MaruyamaDagger , Takanori KumagaiDagger , Yasuyuki MatobaDagger , Minoru HayashidaDagger , Tomomi Fujii§, Yasuo Hata§, and Masanori SugiyamaDagger

From the Dagger  Institute of Pharmaceutical Sciences, Faculty of Medicine, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551 and the § Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

Received for publication, October 30, 2000, and in revised form, December 29, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 beta -turn composed of the residues from Gln97 to Thr102. 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 beta -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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Bleomycin (Bm),1 an antibiotic produced by Streptomyces verticillus, is widely employed in the treatment of several neoplastic diseases including non-Hodgkin's lymphoma, squamous cell carcinomas, and testicular tumors (1, 2). The Bm·Fe(II) complex, in conjunction with a reducing agent and oxygen, causes nucleotide sequence-specific DNA cleavage (3). It has been suggested that "activated Bm," generated by the reductive activation of oxygen by a Bm·Fe(II) complex, cleaves DNA (3, 4). Recent studies have provided unequivocal evidence for Bm-mediated degradation of certain RNA substrates, notably transfer RNAs and tRNA precursor transcripts (5-7).

We have cloned and sequenced a gene, designated blmA, encoding a Bm resistance determinant from Bm-producing S. verticillus (8). The gene blmA encodes a Bm-binding protein, designated BLMA (8, 9). Tallysomycin-producing Streptoalloteichus hindustanus produces a protein designated Shble protein, which binds to Bm like BLMA (10). When incubated with Bm-binding proteins, Bm loses both its antibacterial and DNA-cleaving activities (8, 10).

Although Bm has not been used as an antibacterial agent, almost all strains of methicillin-resistant Staphylococcus aureus, isolated in Hiroshima University Hospital, were resistant to the drug. A Bm resistance gene, designated blmS, has been cloned from chromosomal DNA isolated from methicillin-resistant S. aureus and sequenced (11). The gene product, designated BLMS, is also a Bm-binding protein (9).

The transposon Tn5, expressed in Escherichia coli, carries Bm resistance together with kanamycin and streptomycin resistances (12-14). The nucleotide sequence analysis of the Bm resistance gene, designated ble, has suggested that it encodes a protein consisting of 126 amino acids with a molecular mass of 14,058 daltons (14). In addition to its role as the Bm resistance determinant, a ble product confers survival advantage to E. coli (15, 16). The amino acid sequence of the ble gene product, designated by us as BLMT (17), shares sequence homology with the BLMA protein (21%) and the Shble protein (25%). BLMT has been determined to be a Bm-binding protein (17, 18).

We have determined the crystal structure of BLMA at a high resolution of 1.5 Å by the single isomorphous replacement method including the anomalous scattering effect (19). Another group has determined the crystallographic analysis of the Shble protein at a 2.3 Å resolution (20). Both groups have independently provided a model that suggests that dimeric formation of the protein generates two pockets for binding to Bm. However, because the crystallization of BLMA and the Shble protein, which are complexed with Bm, has been unsuccessful until now, the precise binding mode between the protein and Bm has not been determined.

In this study, we successfully crystallized BLMT uncomplexed and complexed with Bm; the former and latter structures were determined at 1.7 and 2.5 Å resolution, respectively. We describe the conformational differences of BLMT in the Bm-free and Bm-bound form. Although a structural model of Bm bound to an oligonucleotide, determined by two-dimensional NMR analysis, has been proposed (21, 22), we report a structural model based on x-ray crystallography.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Crystal Preparation-- BLMT, overproduced using an E. coli host vector system, was purified according to the methods described previously (17, 23). For crystallization, BLMT was adjusted to a final concentration of 21 mg/ml. Crystals were obtained by vapor diffusion at 25 °C using the hanging-drop method (24) with the mother liquor of 25% PEG 6000 (polyethylene glycol), 0.1 M calcium acetate, and 0.1 M sodium cacodylate at pH 7.0. These crystals belong to the orthorhombic space group C2221 with unit cell dimensions a = 81.3 Å, b = 85.0 Å, and c = 78.8 Å. The crystal volume per unit of mass, VM (25), is 2.42 Å3/Da with 2 BLMT monomers in the asymmetric unit, which corresponds to a solvent content of 49%.

For crystallization of BLMT complexed with Bm, the protein was incubated at a final concentration of 14 mg/ml for 1 h at room temperature with a 3-fold molar excess of Bm A2 sulfate (Fig. 1). The molar ratio of Bm A2 was suitable for complete binding to BLMT. The crystals of the BLMT·Bm complex were obtained by vapor diffusion at 25 °C using the hanging-drop method with the mother liquor of 15% PEG 6000, 0.1 M ammonium sulfate, 0.02 M magnesium chloride, and 0.05 M MES-NaOH at pH 5.0. These crystals belong to the orthorhombic space group P212121 with unit cell dimensions a = 115.3 Å, b = 117.0 Å, and c = 79.9 Å. The VM is 2.14 Å3/Da with four 2:2 complexes (2:2 = two monomeric BLMT molecules complexed with two Bm molecules) in an asymmetric unit. The value corresponds to a solvent content of 42.5%.

Data Collection and Processing-- Diffraction data for the Bm-free BLMT crystals were collected with synchrotron radiation (1.0 Å wavelength) at beam line 18B of the Photon Factory, the National Laboratory for High Energy Physics, Tsukuba, Japan. The data collection was done with three crystals. Three sets of high resolution data up to 1.7 Å resolution were recorded on Fuji imaging plates (400 × 800 mm) using a Sakabe's Weissenberg camera (26). The diffraction images were processed to intensity data with the program WEIS (27). The intensity data from each imaging plate were scaled to each other by the Fox and Holmes method (28) and were then merged.

The diffraction data for a crystal of BLMT complexed with Bm was collected on a Rigaku R-AXIS IIC imaging plate detector system using the mirror monochromated CuKalpha radiation produced by a Rigaku RU-300 rotating anode generator operated at 40 kV and 100 mA. The crystal-to-detector distance was set to 95 mm. Each frame of the 1.5° crystal oscillation was collected for 10 min. The diffraction spots in a rotation range of 90° were recorded on a total of 60 frames. The data processing was accomplished at 2.5 Å resolution with the R-AXIS IIC data processing software package. The number of reflections having adequate intensities was small; this may be because of the noncrystallographic translational symmetry causing the systematic weakness of the intensities. Details of the data collection are summarized in Table I.

Structure Determination and Refinement of BLMT Uncomplexed with Bm-- The crystal structure of BLMT was solved by the molecular replacement method using the programs in X-PLOR (29). The start model was a dimeric BLMA structure previously determined at 1.5 Å resolution (19). However, the N-terminal Met1 and Val2 and the C-terminal Gly121 and Glu122 residues in BLMA were omitted from the model, together with four turn regions (Val20-Trp23, Arg52-Ile57, Trp78-Ala81, and Glu99-Gly103).

Atomic coordinates, obtained by the molecular replacement method, were refined against the data from 10 to 4.0 Å for 20 cycles with the entire monomer as a rigid body, resulting in a crystallographic R-factor of 56.4%. Atomic refinement of the model was performed with the combination of simulated annealing (30) and conventional restrained refinement methods (31) using the program X-PLOR. In lower resolution refinement, strict noncrystallographic symmetry restraint was imposed. Noncrystallographic symmetry restraint, gradually weakened according to the progress of refinement, was finally ignored. A subset of 10% of the reflections was used to monitor the free R-factor (Rfree) (32). Individual B-factors were refined by starting with a mean B-factor of 22 Å2 estimated from the Wilson plot (33).

The refinement was started with data from 10 to 3.5 Å resolution and finished at 2.0 Å resolution. Positional parameters and refinement for individual isotropic B-factors were included in each refinement cycle. The program Xfit in the XtalView suite (34) was used for the visualization and building of the model. In the final stage of the X-PLOR refinement, Rfree and the R-factor were improved to 23.8 and 20%, respectively, against the 18,250 reflections with F > 2 sigma  from 10 to 2.0 Å resolution. Further refinement was performed with the data set from 5.0 to 1.7 Å resolution using the program SHELXL-97 (35). The final R-factor was 19.1% for 26,798 reflections between 5.0 and 1.7 Å resolution.

The current model contains two protein monomers in the asymmetric unit, together with 111 water molecules, two tetraethylene glycol moieties, and one calcium ion. Because Met1 and Leu122-Ser126 were invisible on the electron density due to their higher flexibility, they were excluded from the current model. All side-chain atoms of the residues from Thr2 to Leu121, except Glu98, are included in the model. The side-chain atoms of Glu98 were completely invisible and, above the Cbeta , the atoms of the Glu98 side-chain are excluded from the model. A Luzatti plot (36) revealed that the mean coordinate error was ~0.2 Å. No residues except Trp99 are in the acceptable regions of the Ramachandran plot (37, 38). A peptide bond between Tyr88 and Pro89 is in cis-conformation. The r.m.s. deviations from the ideal values are 0.007 Å in bond length, 2.0° in bond angle, and 1.23° in improper angle (Table I). The average B-factors are 26.3 Å2 and 39.5 Å2 for 1892 all-protein atoms and for 138 non-protein atoms, respectively.

Structure Determination and Refinement of BLMT Complexed with Bm-- Pseudo-precession photographs indicate that the reflection intensities (I (hkl)) were systematically weak if k was an odd number. When calculated using the reflections from 10.0 to 4.0 Å resolution, the second highest peak, which is 6% lower than the origin peak, was present at (x, y, z) = (0, 0.5, 0) on the Patterson map. This suggests that the noncrystallographic translational symmetry parallel to the b-axis is present in the crystal. To obtain the solutions of the rotation and the translation functions by decreasing the number of independent molecules per asymmetric unit, we made modifications as follows: the cell length of b-axis was halved. The axis transformation of the crystalline lattice was performed to satisfy the international rule for orthorhombic crystals (b > a > c). In other words, we assumed that this crystal belongs to the space group P21212 with the unit cell dimensions a = 79.9 Å, b = 115.3 Å, and c = 58.5 Å having two 2:2 complexes in an asymmetric unit. Simultaneously, the data set of the structure factor amplitude (F (hkl)) was transformed as follows: when k is an odd number, the reflection was ignored. Otherwise, if k was an even number, the Miller indices of F (hkl) were transformed as (h'k'l') = (l h k/2), where (h'k'l') are the transformed Miller indices and (hkl) are the original ones. Initially, we solved the structure by the molecular replacement method and refined the atomic parameters using the transformed structure factor amplitudes and cell parameters with the programs in X-PLOR. The Bm-free form of dimeric BLMT was provided as a search model.

The atomic coordinate, obtained by the molecular replacement method, was refined against the data from 10.0 to 4.0 Å with F > 2 sigma  for 20 cycles with the entire monomer as a rigid body, resulting in a crystallographic R-factor of 32.8%. The atomic refinement of the model was performed in the combination of the simulated annealing and the conventional restrained refinement methods, imposing strict noncrystallographic symmetry restraint. The refinement was started with data having 10.0-3.5 Å resolution, and the upper limit was raised to 2.5 Å resolution. The noncrystallographic symmetry restraint was weakened using the Rfree value as an indicator. The program Xfit was used for visualizing and rebuilding the model. In the course of the refinement, because the Fo - Fc map showed that Bm was present in the crystal, we constructed the model of Bm into the difference electron density and included it in the subsequent refinement cycles. Several cycles of X-PLOR refinement resulted in decreasing both the R-factor and Rfree to 18.6 and 24.5%, respectively, for the reflections from 10.0 to 2.5 Å resolution with F > 2 sigma .

For refinement using the original cell parameters, the refined atomic coordinate of two 2:2 complexes was transformed as (x', y', z') = (y, z, x +1/4), where (x, y, z) is the atomic coordinate in the halved crystalline lattice as (x', y', z') is in the original one. The translational term (1/4) is necessary to correct the positional shift of the crystal origin. The atomic coordinates of the adjacent molecules, which are related by the crystallographic translational symmetry parallel to c-axis in the halved crystalline lattice, were transformed according to the above formula. The atomic coordinates of four 2:2 complexes were refined against the data between 10.0 and 3.5 Å with F > 2 sigma  for 20 cycles considering the entire 2:2 complex as a rigid body, resulting in a crystallographic R-factor of 21.7%. Because the number of reflections with adequate intensities was smaller when compared with the number of refined parameters, we paid attention to the following points: the noncrystallographic symmetry restraint imposing on the proteins and Bm was strongly set and decreased according to the Rfree value. After the bulk solvent correction (39) was done, reflections at low resolution together with weak reflections with F < 2 sigma  were used for the refinement. Further cycles of atomic refinement yielded the current model. The R-factor and the Rfree were improved to 22.1 and 30.2%, respectively, against all reflections from 30.0 to 2.5 Å resolution. When limited to the reflections with F > 2 sigma , the final R-factor and the Rfree were 19.0 and 26.4%, respectively.

In the present study, we prepared the original topology and parameter files of the Bm molecule. The metal-binding domain in the Bm A2 molecule is composed of the beta -aminoalanine, pyrimidinyl propionamide, and beta -hydroxyhistidine moieties. The binding domain for DNA is composed of the gamma -aminopropyl dimethylsulphonium and bithiazole moieties (Fig. 1). The parameters used for construction of a three-dimensional model of the former and latter moieties were obtained from the x-ray crystal structures of the P-3A·Cu(II) complex (40) and 3-(2'-phenyl-2,4'-bithiazole-4-carboxamide)propyl dimethylsulfonium iodide (41), respectively. The parameters for threonine and methyl valerate in the linker moiety and for gulose and mannose in the sugar moiety of the Bm molecule were prepared using the energy minimization structure, which is calculated by the program CAChe.


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Fig. 1.   The structure of Bm A2. The junctions between the molecular units comprising Bm A2 are indicated by wavy lines. The underlined N atoms are the putative equatorial ligands to the metal ion.

The current model contains 8 monomeric BLMT molecules, 8 Bm molecules, and 48 water molecules in the asymmetric unit. The monomeric BLMT molecule in the current model consists of the Thr2-Glu120 residues, because both terminal residues, Met1 and Glu121-Ser126, were invisible in the electron density. All of the side-chain atoms of the Thr2-Glu120 residues are included in the model. In the electron density, the gamma -aminopropyl dimethylsulphonium and the beta -aminoalanine moieties of Bm are almost invisible and poorly defined, respectively. The mean coordinate error was estimated from a Luzzati plot to be 0.32 Å. The Ramachandran plot indicates that the backbone torsion angles for 67.9% of the nonglycine residues are in the favor region, whereas those for 95.9% are in the acceptable region. Most of the remaining residues, which fall outside of the acceptable region, have poorly defined electron densities and may have large errors in the parameters. The r.m.s. deviations from the ideal values are 0.007 Å in bond length, 1.2° in bond angle, and 0.57° in improper angle (Table I). The average B-factors are 28.1 Å2 for 7544 all-protein atoms and 44.2 Å2 for the 768 atoms of the Bm molecule.

                              
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Table I
Data collection and refinement statistics

Measurement of Dissociation Constant of BLMT for Bm-- Bm A2 sulfate was added at a range of 0.05 to 0.5 µM to 2 ml of 1 mM Tris-HCl (pH 7.5) containing 0.4 µM BLMT. Experiments were done at 25 °C using a spectrofluorophotometer (model RF-5000, Simadz, Japan) under the condition of lambda  excitation = 280 nm, lambda  emission = 350 nm, slit width at excitation = 10 nm, and slit width at emission = 10 nm. To estimate the concentration of protein contained in solution, the molar absorptivity (epsilon  = 19,800), calculated from the number of Trp and Tyr residues in the BLMT molecule, was employed. The dissociation constant of BLMT for Bm was calculated as described previously (19).

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Overall Structure

The monomeric BLMT molecule consists of two alpha -helices and two beta -sheets (beta 2-beta 5 and beta 6-beta 9), in addition to a short N-terminal beta 1-strand (Fig. 2A). The BLMT monomer exhibits two very similar domain structures connected to a long loop from Pro51 to Trp59. The first domain consists of the alpha 1-helix and four beta -strands (beta 2, beta 3, beta 4, and beta 5). The beta 2-strand is parallel to the beta 5-strand, and other pairs of beta -strands (beta 3 and beta 4, beta 4 and beta 5) are in an anti-parallel configuration. The alpha 1-helix plays a role as a linker connecting the beta 2- and beta 3-strands. Similar topology (beta alpha beta beta beta ) is found in the second domain (alpha 2-helix and beta 6-beta 9 strands). The hydrogen bonding networks of the beta 3- and beta 7-strands to each partner's beta -strand generate an atypical anti-parallel beta -sheet because they are disordered (Fig. 2B).


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Fig. 2.   Topology and hydrogen bonding diagram of BLMT. A, the topology of the BLMT monomer. The red boxes and green arrows indicate the alpha -helix and beta -strand, respectively. BLMT contains two alpha -helices and nine beta -strands. Pro7 twists the beta 1- and beta 2-strands. B, hydrogen bonding pattern involving the main-chain atoms of beta -strands. The hydrogen bonds are represented by arrows.

Two monomeric BLMT molecules are related by a noncrystallographic 2-fold axis (Fig. 3A). The beta 1-strand interacts with the partner's beta 6-strand, suggesting that the former strand plays a key role for the dimeric formation. The topology of BLMT is almost the same as BLMA (19) and the Shble protein (20). The dimer formation of BLMT, generated by the alternate arm exchange of two monomeric BLMT molecules, results in two large concavities and two long grooves.


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Fig. 3.   A ribbon model of BLMT dimer and comparison of the Calpha backbones of two monomers. A, the BLMT dimer is represented as a ribbon drawing viewed along the b axis. Two monomers are colored in red and green. The dimer is associated by the noncrystallographic 2-fold axis. Bm-binding pockets are indicated by yellow arrows. B, superposition of the Calpha backbones of two BLMT monomers. Calpha backbones of two monomers comprising the BLMT dimer are colored in red and green.

Although a noncrystallographic symmetry restraint is not imposed in the final refinement, one monomeric BLMT molecule is almost the same as the partner's monomer. The superposition of main-chain atoms on each BLMT monomer is shown in Fig. 3B. The r.m.s. positional differences between two monomeric BLMT molecules are 0.41 Å for main-chain atoms and 0.79 Å for all-protein atoms. Structural deviations occur at two turn regions, Ala49-Ser58 and Leu96-Thr102, and at a long loop composed of the Gln82-Gly87 residues. The conformational deviations may be generated because of the flexibility of these regions.

The overall structure of BLMT complexed with Bm is almost the same as that of the Bm-free BLMT (Fig. 4). The current model shows with accuracy that two Bm molecules bind to two Bm-binding pockets formed by the alternate arm exchange of two monomeric BLMT molecules.


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Fig. 4.   Stereo view of the BLMT complexed with Bm. Calpha backbones of BLMT dimer are colored in orange. Two Bm molecules are bound to Bm-binding pockets. In the Bm molecule, carbon, nitrogen, oxygen, and sulfur atoms are colored in gray, blue, red, and yellow, respectively.

Binding Mode between Bleomycin and BLMT

The bithiazole moiety of Bm is inserted into the long groove running along the dimer interface (Fig. 5A). The first thiazolium ring, which is interacted by the hydrophobic effect with the Phe30B benzene, is also interacted by the polar effect with the Arg65A guanidino group (Fig. 5B). The second thiazolium ring is tightly stacked with two indole rings from Trp35B and Trp99A (Fig. 5B). This stacking effect contributes to stabilization of the Bm molecule.


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Fig. 5.   Binding mode of Bm to BLMT. A, stereo view of the Bm-binding pockets holding a Bm molecule. The two monomers of BLMT dimer are colored in blue and red. The Bm molecule is in green. The metal-binding, linker, and sugar domains of Bm are buried in the large concavity. The bithiazole and terminal amine moieties of Bm are inserted into the long groove. B, stacking of the bithiazole moiety of Bm with BLMT. The amino acid residues from BLMT are colored in red (monomer A) and blue (monomer B). The Bm molecule is colored in green. The dotted lines indicate hydrogen bonds.

The Trp35B indole ring is interacted by a hydrogen bond with the Glu46B carboxylate. The Glu46B carboxylate, interacted by two salt bridges with the Arg65A guanidino group, is also stabilized by a hydrogen bond with the Thr6B hydroxyl oxygen (Fig. 5B). That is, a hydrogen bonding network is extended from Trp35B to Arg65A. In addition, the Arg65A and Trp35B are interacted with the first and second thiazolium rings, respectively.

The large concavity is formed mainly by the residues from one monomer. One side of the concavity has the hydrophobic residues. The linker, metal-binding, and sugar domains of Bm are buried in the large concavity (Fig. 5A) and stabilized by a large number of hydrogen bonds to the protein atoms.

In the metal-binding domain of Bm, an amino group attached to the pyrimidine ring is interacted by a hydrogen bond with two backbone carbonyl oxygens of Phe60A and Gly111A (Fig. 6). The carbonyl oxygen of the pyrimidinyl propionamide moiety of Bm forms a hydrogen bond with the Arg90A guanidino group (Fig. 6). The terminal amide group of the pyrimidinyl propionamide moiety forms three hydrogen bonds: its oxygen atom with the Arg115A guanidino group, its nitrogen atom with the Ser61A hydroxyl oxygen, and the Trp59A carbonyl oxygen (Fig. 6). However, beta -aminoalanine and beta -hydroxyhistidine for the metal binding make no polar interactions with the protein atoms. Because the beta -aminoalanine moiety is poorly defined in the electron density, the conformation may be unstable. However, its amide group orients parallel to the Trp59A indole ring and seems to be stacked by the apolar interaction. In fact, the Trp59A side-chain is flexible in the Bm-free form but rigid in the complexed form because the side-chain is stacked with the imidazole rings of His50B and the amide group of the beta -aminoalanine moiety of Bm.


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Fig. 6.   Hydrogen bonding network of Bm to BLMT in the large concavity. The residues from Bm and BLMT are colored in black and gray, respectively. The oxygen and nitrogen atoms are colored in red and blue, respectively. The dotted lines indicate hydrogen bonds.

Only mannose, but not gulose, in the sugar moiety of Bm is involved in the binding to the protein. The carbamoyl group, attached to the 3' hydroxyl oxygen of mannose, makes two hydrogen bonds: its nitrogen atom with the carbonyl oxygens of Pro55A and Ser58A (Fig. 6). The 2' and 4' hydroxyl groups form a hydrogen bond with the Leu56A carbonyl oxygen and the Arg90B guanidino group, respectively. The 6' hydroxyl group interacts by the hydrogen bond with the Arg90B guanidino, Ser85B carbonyl, and Gly87B amide groups (Fig. 6).

The interaction between the protein and the linker domain of Bm is weak. Only one hydrogen bond is formed between the Arg115A guanidino group and the carbonyl oxygen of the threonine moiety of Bm (Fig. 6). This occurrence is likely to be related with the flexibility of the domain.

The terminal gamma -aminopropyl dimethylsulphonium moiety of Bm A2, having the positive charge, is not well defined in the electron density. The end of the long groove, which is the expected binding site for the gamma -aminopropyl dimethylsulphonium moiety, has negatively charged residues Asp3, Asp42, Asp67, and Glu120. These residues are located around the positive charge of the gamma -aminopropyl dimethylsulphonium moiety. However, no electrostatic interactions are observed between this moiety and the negatively charged residues, suggesting that the negatively charged residues are necessary for the recognition of the ligand rather than for stabilization.

Conformational Change of BLMT by Binding of Bm

Fig. 7A shows the superposition of the BLMT·Bm structure on the Bm-free structure. The positional r.m.s. differences between two structures are 0.53 and 0.77 Å for the main-chain atoms and all-protein atoms, respectively. The marked positional deviation occurs at the residues located on the loop and turn regions, such as His50-Ala57, Glu83-Gly87, and Gln97-Thr102. In particular, the Gln97-Thr102 residues, composed of a beta -turn between the beta 7- and beta 8-strands, move about 2 Å toward the partner's monomer. The conformational change enables the Trp99 indole ring of one monomer to approach the Trp35 indole ring of the partner's monomer. The conformational approach results in the formation of stable structure of the long groove and strong intercalating interaction with the bithiazole moiety of Bm. However, the corresponding region of the Bm-free BLMT appears to be highly flexible with large B-factors (Fig. 7B).


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Fig. 7.   Conformational change of BLMT by binding to Bm. A, superposition of BLMT uncomplexed with Bm onto that complexed with Bm. Calpha backbones of BLMT uncomplexed and complexed with Bm are colored in red and blue, respectively. B, B-factor of BLMT uncomplexed and complexed with Bm. The black and red lines indicate the average B-factor for main-chain atoms of BLMT uncomplexed and complexed with Bm, respectively.

Comparison with Bm-binding Proteins from Other Microbial Sources

Overall Structure-- The amino acid sequence homology between BLMA and the Shble protein is ~60%, whereas that between BLMT and BLMA, or between BLMT and the Shble protein is only 21-25%. Nevertheless, the superposition of the main-chain atoms of BLMT on that of BLMA or the Shble protein shows that the overall structure of BLMT is almost the same as those of BLMA and the Shble protein (Fig. 8A). The r.m.s. positional differences for 114 residues of the core region composed of beta -sheets are 1.25 Å between BLMT and BLMA and 1.35 Å between BLMT and the Shble protein. The marked structural deviation found among three Bm-binding proteins occurs away from the gravity center of dimeric molecules.


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Fig. 8.   Comparison of BLMT with other Bm-binding proteins. A, superposition of three Bm-binding proteins. Calpha backbones of BLMT, BLMA, and Shble proteins are colored in green, blue, and red, respectively. B, alignment of Bm-binding proteins from different microbial sources. Red and blue indicate acidic and basic residues, respectively. Purple and green arrowheads indicate an amino acid deletion and insertion in BLMT, respectively.

The conformation of a long loop connecting alpha 2-helix and the beta 7-strand in BLMT is more stable than that in BLMA or the Shble protein. The stability is likely to be generated by the deletion of one amino acid in this region (Fig. 8B). Otherwise, the turn region between the beta 7- and beta 8-strands of BLMT is highly flexible. The flexibility may be generated by the insertion of an amino acid into the long loop (Fig. 8B).

Substrate Specificity-- Table II lists the dissociation constants (Kd) to Bm and phleomycin (Phm) for BLMT and other Bm-binding proteins. Phm is an analog of Bm and has a thiazolinylthiazole instead of a bithiazole moiety in Bm; that is, the second thiazolium ring in Phm is reduced and nonplanar. We have observed that the affinity between BLMT and Phm is stronger than between BLMA and Phm. Although the Kd value to Phm for the Shble protein has not been determined yet, there is a report that the minimum inhibitory concentration of Phm to E. coli expressing the Shble protein is much lower than that of Bm (20). This suggests that the binding affinity of the Shble protein to Phm is lower than to Bm. The difference of the dissociation constant between Bm and Phm can be explained as follows. The turn region of BLMT composed of the Gln97-Thr102 residues, which is the Bm-binding domain, is more flexible than those of BLMA and the Shble protein. The flexibility of the binding domain in BLMT is generated by the insertion of an additional amino acid into the turn region (Fig. 8B). The flexibility might decrease the substrate specificity for binding.

                              
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Table II
Ligand specification of Bm-binding proteins

Bm-binding Sites-- Aromatic residues corresponding to Phe30 and Trp35 of BLMT, which interact with the bithiazole moiety of Bm, are present in BLMA, BLMS, and the Shble protein (Fig. 8B), suggesting that the aromatic rings are interacted by the hydrophobic effect with the bithiazole moiety. An amino acid corresponding to Trp99 of BLMT, which interacts with the bithiazole moiety of Bm, is also present in BLMS and the Shble protein. The Trp residue of these proteins is likely to interact with the bithiazole moiety. BLMA is replaced by Ala instead of Trp99. A crystallographic analysis of BLMA complexed with Bm showed that Pro101 interacts with the bithiazole moiety.2

The negatively charged residues of the BLMT·Bm complex are located around the positive charge of the gamma -aminopropyl dimethylsulphonium moiety of Bm A2, but no electrostatic interactions are found between the protein and Bm. The distribution of the negatively charged residues in BLMT (Fig. 8B) resembles those in other Bm-binding proteins (19, 20). These negatively charged residues might be helpful in the recognition of the ligand.

Structure of Bleomycin

Despite the powerful investigation of the x-ray crystal structure of Bm for the last two decades, the structure has not yet been determined. Therefore, the controversial and elusive points on the structure of the Bm molecule must be resolved; for example, it is not yet clear whether the two thiazolium rings in the bithiazole moiety of the Bm molecule are in cis- or trans-conformation. The crystallographic study of bithiazole derivatives has suggested that the two thiazolium rings may be in trans-conformation (41, 42). Some groups have determined that the solution structure of bithiazole moiety bound to DNA and suggested that the cis-conformation is favored to account for the upfield shift of the chemical shift of the bithiazolium ring protons (43, 44). In the present study, we tried to model both cis- and trans-type bithiazole moiety. From the electron density map obtained in this study, we conclude that the two thiazolium rings are in trans-conformation.

The ligands of the Bm molecule for the metal ion and its chirality also remain ambiguous. Most investigators agree on the fact that the equatorial ligands are the secondary amine of the beta -aminoalanine moiety, the amide nitrogen of the beta -hydroxyhistidine moiety, and the nitrogens from the pyrimidine and the imidazole rings. However, three possibilities are emphasized for the axial ligand: (i) the primary amine of the beta -aminoalanine moiety (45-47); (ii) the carbamoyl nitrogen of the mannose moiety; and (iii) the primary amine of the beta -aminoalanine moiety and the carbamoyl nitrogen of the mannose moiety (48-50). Although our model of BLMT complexed with Bm does not contain the metal ion, we suggest that the primary amine of the beta -aminoalanine moiety is suitable as an axial ligand for the metal ion. In the Bm molecule, while the carbamoyl group of the mannose moiety takes a stable conformation by forming the hydrogen bonds to the protein atoms, the conformation of beta -aminoalanine moiety is unstable because of the lack of polar interactions with the protein atoms. Furthermore, the primary amine of the beta -aminoalanine is just positioned over the putative equatorial plane for the metal ion with the same chirality as the propositions (45-47). Judging from these observations, the primary amine of the beta -aminoalanine moiety may be an axial ligand.

The most interesting aspect in the research of the structure of Bm is the interpretation of the DNA-cleavage activity of Bm. Until now, models of Co(III)·Bm A2 complexed with DNA have been built by NMR analysis (21, 22). In the model, the bithiazole moiety is inserted into the space between two base pairs and stacked with the pyridine and pyrimidine rings. The positively charged terminal amine of Bm is buried in the major groove of DNA and is interacted with the negatively charged backbone phosphate. The linker and metal-binding domains of Bm, located in the minor groove of DNA, are stabilized by several hydrogen bonds with DNA including base pair-specific ones.

The binding mode of Bm complexed with BLMT may be similar to that with DNA for the following reasons. First, the bithiazole moiety is intercalated with the aromatic rings in both cases of DNA and BLMT. Second, the linker and metal-binding domains are buried in the minor groove of DNA or the large concavity of BLMT and stabilized by a number of hydrogen bonds. Finally, the positive charge of the terminal amine of Bm may be necessary for the electrostatic interaction with DNA or BLMT.

A model of the DNA·Bm complex shows that Bm exhibits a more compact form, that is, the bithiazole moiety folds back toward the metal-binding domain (21, 22). However, x-ray analysis of Bm complexed with BLMT shows that the Bm molecule has a more extended form, that is, the bithiazole moiety is far from the metal-binding domain.

    ACKNOWLEDGEMENTS

We thank Dr. Y. Kawano (RIKEN Harima Institute, Japan) for valuable discussion on x-ray crystallographic analysis and for kind help in data collection at the Photon Factory (Tsukuba, Japan). We are grateful to T. Miyazaki (Nippon Kayaku Co., Ltd.) for the gift of bleomycin A2 sulfate.

    FOOTNOTES

* This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1ecs for BLMT uncomplexed with bleomycin (Bm) and code 1ewj for BLMT complexed with Bm) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

To whom correspondence should be addressed. Tel.: +81-82-257-5280; Fax: +81-82-257-5284; E-mail: sugi@hiroshima-u.ac.jp.

Published, JBC Papers in Press, December 29, 2000, DOI 10.1074/jbc.M009874200

2 M. Sugiyama, M. Hayashida, Y. Matoba, M. Maruyama, and T. Kumagai, unpublished data.

    ABBREVIATIONS

The abbreviations used are: Bm, bleomycin; BLMT, a bleomycin resistance determinant from the transposon Tn5; BLMA, blmA gene product from Streptomyces verticillus ATCC 15003 (Bm producer); Shble, a gene encoding a Bm-binding protein from Streptoalloteichus hindustanus (tallysomycin producer); BLMS, the protein encoded by blmS from Bm-resistant methicillin-resistant Staphylococcus aureus B-26; MES, 2-(N-morpholino)ethanesulfonic acid; r.m.s., root mean square; Phm, phleomycin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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