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J. Biol. Chem., Vol. 276, Issue 13, 9992-9999, March 30, 2001
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From the
Received for publication, October 30, 2000, and in revised form, December 29, 2000
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 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.
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 CuK 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
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 C 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
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
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
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 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 Overall Structure
The monomeric BLMT molecule consists of two Two monomeric BLMT molecules are related by a noncrystallographic
2-fold axis (Fig. 3A). The
Crystal Structures of the Transposon Tn5-carried
Bleomycin Resistance Determinant Uncomplexed and Complexed with
Bleomycin*
,,,,¶
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
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-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
-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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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.
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 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.
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 .
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 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 , the final R-factor and the
Rfree were 19.0 and 26.4%, respectively.
-aminoalanine,
pyrimidinyl propionamide, and -hydroxyhistidine moieties. The
binding domain for DNA is composed of the 
-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.
-aminopropyl dimethylsulphonium
and the -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.
Data collection and refinement statistics
excitation = 280 nm, 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 (
= 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helices and two
-sheets (2-5 and 6-9), in addition to a short
N-terminal 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 1-helix and four -strands (2, 3, 4, and 5). The
2-strand is parallel to the 5-strand, and other pairs of
-strands (3 and 4, 4 and 5) are in an anti-parallel
configuration. The 1-helix plays a role as a linker connecting the
2- and 3-strands. Similar topology () is found in
the second domain (2-helix and 6-9 strands). The hydrogen bonding networks of the 3- and 7-strands to each partner's
-strand generate an atypical anti-parallel -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 -helix
and -strand, respectively. BLMT contains two -helices and nine
-strands. Pro7 twists the 1- and 2-strands.
B, hydrogen bonding pattern involving the main-chain atoms
of -strands. The hydrogen bonds are represented by
arrows.
1-strand interacts with the partner's 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.
View larger version (20K):
[in a new window]
Fig. 3.
A ribbon model of BLMT dimer and comparison
of the C 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 C backbones of two BLMT monomers. C 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.
|
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.
|
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, -aminoalanine
and -hydroxyhistidine for the metal binding make no polar
interactions with the protein atoms. Because the -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 -aminoalanine moiety of Bm.
|
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 -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 -aminopropyl dimethylsulphonium moiety, has
negatively charged residues Asp3, Asp42,
Asp67, and Glu120. These residues are located
around the positive charge of the -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 -turn
between the 7- and 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).
|
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 -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.
|
The conformation of a long loop connecting 2-helix and the
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 7- and 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.
|
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 -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 -aminoalanine
moiety, the amide nitrogen of the -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 -aminoalanine moiety (45-47); (ii) the
carbamoyl nitrogen of the mannose moiety; and (iii) the primary amine
of the -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 -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 -aminoalanine moiety is unstable
because of the lack of polar interactions with the protein atoms.
Furthermore, the primary amine of the -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 -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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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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.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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