The 1.5 Å crystal structure of a bleomycin resistance determinant from bleomycin-producing Streptomyces verticillus1

Article No. jmbi.1999.3404 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 295, 915±925

Ê Crystal Structure of a Bleomycin Resistance The 1.5 A Determinant from Bleomycin-producing Streptomyces verticillus Yoshiaki Kawano1, Takanori Kumagai2, Kengo Muta2 Yasuyuki Matoba2 Julian Davies3 and Masanori Sugiyama2* 1

RIKEN Harima Institute, Mikazuki-cho, Sayo, Hyogo 679-5143, Japan 2 Institute of Pharmaceutical Sciences, Faculty of Medicine Hiroshima University Hiroshima, 734-8551, Japan 3

Department of Microbiology and Immunology, The University of British Columbia 300-6174, University Boulevard, Vancouver, BC Canada V6T 1Z3

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 Ê with a merging intensity data were collected up to a resolution of 1.5 A R-value of 0.054 at a completeness of 94 %. The BLMA structure, determined by the single isomorphous replacement method including the Ê , was re®ned anomalous scattering effect (SIR-AS) at a resolution of 2.0 A Ê resolution. The ®nal R-factor was 19.0 % and Rfree was 22.1 % at 1.5 A including 91 water molecules. The crystal packing showed a dimer Ê high-resolution form, which was generated by arm exchange. The 1.5 A 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 Ser100Gly103 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 (K1/2) of Bm is higher for BLMA than for the Shble protein. # 2000 Academic Press

*Corresponding author

Keywords: bleomycin; bleomycin-binding protein; X-ray crystallography; arm-exchange; Streptomyces verticillus

Introduction Bleomycin (Bm), produced by Streptomyces verticillus, is used as an important anti-tumor antibiotic, which causes cell death as a result of multiple strand scissions of mammalian and bacterial DNA (Umezawa, 1974). The Fe(II)-Bm complex, in conjunction with reducing agent and oxygen, causes nucleotide sequence-speci®c DNA cleavage (Stubbe & Kozarich, 1987). The DNA cleavage mechanism by Bm has been extensively studied and it has been shown that ``activated Bm'', generated by reductive activation of oxygen by Fe-Bm, is involved in the DNA cleavage process (Stubbe & Abbreviations used: Bm, bleomycin; BLMA, a Bmbinding protein; S., Streptomyces; SIR-AS, the single isomorphous replacement method including anomalous scattering effect; MRSA, methicillin-resistant Staphylococcus aureus. E-mail address of the corresponding author: [email protected] 0022-2836/00/040915±11 $35.00/0

Kozarich, 1987). Early studies reported that RNA was not degraded by Bm, whereas more recent studies have provided unequivocal evidence for Bmmediated degradation of certain RNA substrates, notably tRNAs and tRNA precursor transcripts (Magliozzo et al., 1989; Carter et al., 1990; Holmes et al., 1993). The antibiotic-producing microorganism must be protected from the lethal effect of its own antibiotic product. We have cloned a Bm resistance gene, designated blmA, from Bm-producing S. verticillus ATCC15003 (Sugiyama et al., 1994). The gene product, designated BLMA, has been physico-chemically characterized; it is an acidic protein with a strong af®nity for Bm (Sugiyama et al., 1994, 1995). We have cloned a gene, blmS, conferring resistance to Bm from the chromosomal DNA of a methicillin-resistant Staphylococcus aureus B-26 (Bhuiyan et al., 1995). The blmS gene product, designated BLMS was, like BLMA, a Bm-binding protein (Sugiyama et al., 1995). A gene, ble, located # 2000 Academic Press

916 on the transposon Tn5 (Genilloud et al., 1984; Mazodier et al., 1985), which was originally found as an R-factor of Klebsiella (Berg et al., 1975), also confers Bm resistance to Escherichia coli. We have found that the ble gene product, designated BLMT, is a Bm-binding protein (Kumagai et al., 1999a). Tallysomycin-producing Streptoalloteichus hindustanus, which belongs to the family Pseudonocardiaceae and is distinct from Streptomycetaceae, expresses a Bm-binding protein encoded by a gene Shble (Stackebrandit et al., 1997; Gatignol et al., 1988). A polyclonal antibody, raised against the Shble protein, did not cross-react with BLMA (Sugiyama et al., 1995). A monoclonal antibody, generated against BLMA, also did not cross-react with the Shble protein (Sugiyama et al., 1995). Thus BLMA is imnunologically different from the Shble protein. The crystal structure of the Bm-free Shble protein Ê and has been determined at a resolution of 2.3 A used to make the putative binding model with Bm (Dumas et al., 1994). However, the atomic coordinates for the Shble protein were not deposited in the Protein Data Bank (PDB) until October 17, 1998. In order to ®nd the structural difference between BLMA and the Shble protein, we have obtained the X-ray diffraction data of the native BLMA crystal and determined its structure using Ê , prior to the SIR-AS method at a resolution of 2.0 A the deposition of the Shble protein coordinates in the PDB (Kawano et al., 1996; Kumagai et al., 1998). The present study shows in detail the the crystal structure of BLMA re®ned at high resolution, Ê , including its side-chain disorder and water 1.5 A molecules contributing to dimerization. Furthermore, we build a binding model between Bm and BLMA based on the precise crystal structure of BLMA, and discuss the correlation between the binding af®nity for Bm and the three-dimensional structure in BLMA and the Shble protein.

Results and Discussion Topology of BLMA The monomeric form of BLMA consists of three ahelices (a1, a2 and a3) and two b-pleated sheets, in addition to a short b-strand (b1) twisting at its amino-terminal Pro9 (Figure 1). The BLMA monomer has two very similar domains connected to the a2-helix. The ®rst domain is composed of the a1helix and four b-strands (b2-b5). The four strands form one b-pleated sheet in which the b2 and b5 strands are in a parallel con®guration, and the b3, b4 and b5 strands are in an anti-parallel con®guration. The second domain consists of the a3-helix and ®ve b-strands (b6-b10). The ®ve strands form another bpleated sheet, which is very similar to that in the ®rst domain, except that a strand corresponding to b3 is separated into two parts (b7 and b8). The bstrand structure is broken from Thr95 to Gly98 and this part is not connected with the b9-strand by a hydrogen bond (Figure 2). All components are con-

Crystal Structure of a Bleomycin-binding Protein

nected by short and long loops. While most of the loops are well de®ned, a long loop from Ala79 to Pro92 and a short loop from Ser100 to Gly103 have large temperature factors (Figures 1 and 3). BLMA forms a dimeric structure by alternate arm exchange (Bergdoll et al., 1996) of the monomeric BLMA molecule (Figure 4). Two BLMA monomers are related by a crystallographic 2-fold axis (Figure 4). The N-terminal b1-strand consisting of eight amino acid residues from Met1 to Val8 of the BLMA monomer is connected with its partner's b6strand by hydrogen bonds (Figure 5). The armexchanged b1-strand plays an important role in forming the dimeric structure (Bergdoll et al., 1996; Kumagai et al., 1999b). The protein topology of BLMA is very similar to that of the Shble protein. The dimeric form, generated by alternate arm exchange of the monomeric BLMA molecule, results in two large concavities and two long grooves.

Side-chain disorder Ê ) allowed us to High-resolution analysis (at 1.5 A analyze the side-chain disorder of BLMA. The sidechains of Lys31, Arg36, Ile66, Glu105 and Thr119 are clearly disordered in the crystal structure. The side-chain nitrogen atom of Lys31 in one conformation is connected with the main-chain carbonyl oxygen of Val97 and Wat261 by hydrogen bonds. The side-chain nitrogen atom of Lys31 in the other conformation is connected with the main-chain carbonyl oxygen atom of Val97 and Wat246. The occupancies of the two disordered structures are 0.46 and 0.54, respectively. In the other Bm-binding proteins, listed in Figure 6, Lys31 is not conserved. The side-chain of Arg36 protrudes on the surface of the molecule. The side-chain nitrogen atoms of Arg36 at one conformation makes two salt-bridges with two side-chain oxygen atoms of Asp70. A side-chain nitrogen atom of Arg36 in the other conformation makes a salt-bridge with the sidechain oxygen atom of Asp72, and the other nitrogen atom interacts with Wat261 by a hydrogen bond. The side-chain of Ile66 rotates in a hydrophobic pocket in the monomer. This hydrophobic pocket is present in the hydrophobic cluster, which stabilizes the monomer structure. In the other Bm-resistance determinants (Figure 6), the corresponding residue is Ile, Val or Leu, which have similar side-chains. The residues around Ile66 (Ala7, Leu48 and Val116) in the related proteins also have similar side-chain lengths. Therefore, other Bm-binding proteins may have small related hydrophobic pockets. A side-chain oxygen atom of Glu105 in one conformation makes a hydrogen bond with Nd1 of His117, and the other oxygen atom of the same conformation is connected to Wat295. The side-chain oxygen atoms of Glu105 in the other conformation are not connected with any other atom. The disordered structure on Thr119 indicates the side-chain rotates only around the Ca-Cb bond. The two residues Glu105 and Thr119 on the sur-

917

Crystal Structure of a Bleomycin-binding Protein

Figure 1. Topology diagram of the BLMA monomer. The boxes and green arrows indicate a-helix and b-strands, respectively. BLMA contains three a-helices and ten bstrands. Pro9 twists the b1 and b2strands.

face of the protein are located in a loop consisting of Trp78-Gly98. Water molecules contributing to dimerization Some water molecules are located at the inside of the dimer: of these, three contribute to dimerize BLMA monomers (Figure 5). A hydrogen bonding network is formed by one water molecule (Wat291) with the main-chain nitrogen atom of Val10 in one monomer and the main-chain oxygen atom of Ser63 in the partner's monomer. This water molecule (Wat291) is closely linked to the 2-fold axis

and makes a hydrogen bond with the symmetryrelated water molecule (Wat2910 ). These hydrogen bonding networks connect the b2 and b6-strands. Another hydrogen bonding network is formed by one water molecule (Wat207) with the main-chain oxygen atom of Ala7 and the main-chain nitrogen atom of Arg47 in the same monomer. These three water molecules contribute to ®x the extension direction of the arm-exchanged b1-strand, and support the arm-exchange structure. Another ®ve water molecules (203, 202, 209, 2020 and 2030 ) form a path through the BLMA dimer at the interface of two monomers (Figure 7). The BLMA dimer has a

Figure 2. Stereo-view of hydrogen-bonding network of main-chain between Pro92 Ala120. The hydrogen bonds represented as broken lines.

the the and are

918

Crystal Structure of a Bleomycin-binding Protein

Figure 3. B-factor of BLMA. Continuous and broken lines indicate the average for main-chain atoms (N, CA, C, O) and for side-chain atoms, respectively. The B-factor of the main-chain atoms in N and C termini, Glu76-Ala93 and Val97-Glu105 is larger than that of the other main-chain atoms.

water tunnel from one side to another. The entrance of this tunnel is located on the bottom of the large concavity near Ser63 and Asn61. Molecular surface Figure 8 represents the molecular surface of the BLMA dimer. Many of the charged residues are located on the edge of the large concavity and a long straight groove running from the concavity. Some hydrophilic residues are gathered in the bottom of the large concavity. A hydrophobic region is located in the center of the long groove, in particular, the side-chains of Phe33 and Phe38 are on the molecular surface at the bottom of the groove. In other Bm-binding proteins, such as BLMT and BLMS, Trp or His is present as an aromatic sidechain instead of Phe (Figure 6). The end of the long groove in the BLMA dimer has negatively charged residues such as Asp32, Asp45, Glu67 and Glu122 as seen in the Shble protein. On the contrary, highly conserved amino acids are absent in the concavity and groove regions. Furthermore, the distribution of charged amino acids in these regions is different in each of the Bm-binding proteins. A model of the BLMA/bleomycin complex The BLMA/Bm complex has not been crystallized. In addition, the crystal structure of Bm has not been determined. Therefore, to build the BLMA/Bm complex model, we tried to construct a three-dimensional model of Bm: the information on the iron-ligand part of Bm was obtained from the X-ray crystal structure of P-3A (Iitaka et al., 1978). Both the bond length and the bond angle of the bithiazole ring were taken from the X-ray crystal structure of a bithiazole analog (Kuroda et al., 1995). The program Insight II (Biosym Co. ltd.)

Figure 4. A ribbon model of the BLMA dimer viewed along the b-axis. Two monomers are indicated in red and green. The dimer is associated by the crystallographic 2-fold axis. Two large concavities are indicated by two black arrows.

was used in the preparation of other components of Bm and its whole structure (Figure 9). Dimer formation of BLMA generates two Bmbinding pockets composed of two large concavites and long grooves (Figure 4). One Bm molecule seems to enter into one of the two Bm-binding pockets. The Bm-binding mode in the constructed complex is as follows. The metal-binding site and sugar moiety of Bm are assumed to be buried in the large concavity, since the size of these parts is consistent with that of the concavity (Figure 10(b)). These parts might be stabilized by polar interactions with several hydrophilic amino acid residues that protrude toward the concavity. The tetrapeptide as linker region, and the bithiazole and terminal amine moieties in Bm are inserted into the long groove running at the dimer interface. The overall Bm molecule seems to have an extended form rather than a folded form, as shown in Figure 10(b). The bithiazole ring may attach to the hydrophobic region of the BLMA groove, and stack with benzenoid moieties of Phe33 and Phe38 as shown in Figure 10(a). This stacking may contribute to stabilizing the Bm molecule. The positively charged terminal amine moiety of Bm is located at the end of the long groove and is surrounded by the negatively charged Asp32, Arg42, Asp45, Arg47, Glu67 and Glu122. Thus the terminal amine moiety of Bm may be stabilized by electrostatic force. Although the positively charged residues, such as Arg42 and Arg47, are located in this region, these residues can form salt-bridges with the negative side-chains. Arg42 and Arg47 can connect with Asp32 and/or Glu30, and with Asp32 and/or Glu67, respectively.

Crystal Structure of a Bleomycin-binding Protein

919

Figure 5. A stereo-view of the hydrogen-bonding network. Each monomer is indicated in a series of red or green. The hydrogen bonds are represented as broken lines. The red spheres indicate water molecules that contribute to the dimer formation.

Wu et al. (1996) and Vanderwall et al. (1997) have reported the detailed solution structure of Co-Bm complexed with DNA, which was obtained by two-dimensional NMR experiments. In the model, the bithiazole moiety is partially intercalated between base-pairs. The terminal thiazole ring of Bm is completely stacked with purine bases of DNA, while the penultimate thiazole ring is only partially stacked with pyrimidine bases. The stacking mode of bithiazole moiety with BLMA in our model (Figure 10(a)) is related to the NMR model. The metal-binding site is present within the minor groove of DNA and may be stabilized by a number of hydrogen bonds in their model. The metal-binding region in our model is buried in the

large concavity, which thus appears to correspond to the minor groove of DNA. The binding mode between BLMA and Bm may be similar to that between DNA and Bm. Conformational change of BLMA for binding to Bm Ile66, a disordered residue in BLMA, is positioned at the back side of the b6-strand, which is placed in the bottom of the large concavity. When Bm is held in the hydrophobic pocket near Ile66, the b6-strand might shift a few aÊngstroÈm units to the inside of the BLMA molecule. This would cause the large concavity to become more deeper

Figure 6. Amino acid sequences of Bm resistance determinants from different microbial sources. BLMA, blmA gene product from Streptomyces verticillus ATCC15003 (Bm producer); ShbleP, a Bm-binding protein from Streptoalloteichus hindustanus (tallysomycin producer) (Drocourt et al., 1990); SvP, a Bmbinding protein from S. verticillus ATCC 21890 (phleomycin producer) (Calcutt & Schmidt, 1994); BLMS, the protein encoded by blmS from a Bm-resistant MRSA B-26 (Sugiyama et al., 1995); BLMT, a Bm determinant encoded on the transposon Tn5 (Genilloud et al., 1984; Kumagai et al., 1999a,b): * indicates the amino acid residue on BLMA contacting Bm.

920

Crystal Structure of a Bleomycin-binding Protein

Comparison of BLMA with the Shble protein Overall structure The b-strand in the Shble protein corresponding to the b7 and b8-strands, which are discontinuous in BLMA, was represented as a single strand in a Figure by Dumas et al. (1994). However, from the atomic coordinates deposited in the PDB in 1998, the b-strand is obviously discontinuous, like BLMA, suggesting that the topology of BLMA is very similar to that of the Shble protein. Superposition of BLMA on the Shble protein reveals that these structures are strikingly similar, especially at the core region, which consists of b-sheets. The r.m.s. positional difference between BLMA and the Shble protein for all main-chain Ê , but 0.68 A Ê for the main-chain atoms is 1.13 A atoms of 114 amino acid residues in the core region. Thus, the structural difference between these proteins is relatively large in the loop and helical regions. The Ser100-Gly103 loop in BLMA is farther from the groove, which is the Bm-binding site, than that (Gln100-Gly103) in the Shble protein. These structural differences between the proteins may be one of the reasons that the binding af®nity of Bm is lower for BLMA than that for the Shble protein, as discussed below. Binding affinity between Bm and BLMA

Figure 7. Hydrogen bond network around the water tunnel. The hydrogen bonds are shown by broken lines. Two monomers are indicated in red or blue.

than that in this Bm-free model. This structural change of the b6-strand might contribute to the ®tting of Bm into the large concavity. The anti-parallel b-sheet, formed with b7, b8 and b9, is markedly bent at Thr95-Gly98 and Arg104 because of the rotation of the Pro96 torsion angle. By adopting this structure, the Glu99-Arg104 loop may approach the groove region. When Bm is held in the groove region, the main-chain torsion angles of Arg104-Glu105 and Thr95-Gly98 would be changed to make the groove be in an open or closed state by conformational change of the Glu99Gly103 loop, which might contribute to the binding of Bm to the groove region. This prediction is supported by the ®nding that the Glu99-Arg104 temperature factor is larger in this structure (Figure 3).

A half-saturating concentration (K1/2) of Bm for BLMA, estimated using the ¯uorescence quenching method, was 280 nM, showing that the Bmbinding af®nity of BLMA is lower than that of the Shble protein (K1/2 ˆ 55 nM) (Gatignol et al., 1988). This observation can be explained based on the structural difference between these proteins as follows: (i) strong cofacial stacking interactions are found between two phenyl groups of Phe33 and Phe38 residues in BLMA and the bithiazole ring of the Bm molecule. In the Shble protein, however, amino acid residues stacking with the bithiazole ring have been suggested to be an indole ring of Trp102 in the Gln100-Gly103 loop and the phenyl group of Phe33 (Dumas et al., 1994). The Shble protein has the amino acid identical with Phe38 in BLMA, whereas Phe38 in the former protein has never been discussed for stacking with the bithiazole ring (Dumas et al., 1994). It is reasonable to infer that Phe38 in the Shble protein also stacks with the bithiazole ring of Bm, in addition to Phe33 and Trp102. Signi®cantly, Trp102 in the Shble protein is replaced by Ala in BLMA (Figure 6). This replacement might explain why the stacking ability with Bm is weaker in BLMA than in the Shble protein. (ii) A Ser100-Gly103 loop is farther from the groove in BLMA than the corresponding loop (Gln100-Gly103) in the Shble protein. The loop is suggested to orient toward the groove for binding to Bm. The loop in the Shble protein does not need to turn more towards the groove than that in BLMA in order to bind

921

Crystal Structure of a Bleomycin-binding Protein

Figure 8. Surface of BLMA dimer. Side-chain atoms of acidic, basic, hydrophobic and hydrophilic residues are colored red, blue, yellow and green, respectively. Main-chain atoms are indicated as white. The 2-fold axis runs from top to bottom in this Figure. This Figure, viewed along the arrow direction of Figure 4, was prepared using the GRASP program (Nicholls et al., 1991).

Bm, suggesting that the Shble protein may have the thermodynamic advantage over BLMA. (iii) The hydrophobicity of the groove in BLMA is much lower than that in the Shble protein. For example, the positively charged side-chain of Arg47 in BLMA is much more oriented towards the groove than that of Thr47 in the Shble protein. Thus, hydrophobic interactions between the groove and linker parts of Bm may not be tighter in BLMA than in the Shble protein.

Materials and Methods Crystallization and data collection Crystallization was achieved at 20  C using the hanging drop method (McPherson, 1982) with a drop that contained 5 ml of protein solution at 20 mg/ml and 5 ml of the reservoir solution contained 0.2 M ammonium acetate, 0.1 M sodium acetate (pH 5.7) and 30 % PEG4000. Crystals of up to 0.2 mm  0.2 mm  0.5 mm were obtained after one week; they belonged to the

Figure 9. The structure of bleomycin A2. The junctions between the molecular units comprising Bm A2 are indicated by dotted lines. The underlined N atoms are the putative ligands to the metal according to the crystallographic structure of the Cu(II)-P3A complex (Iitaka et al., 1978).

922

Crystal Structure of a Bleomycin-binding Protein

Figure 10. (a) A stereo-view of Phe33 and Phe38 of BLMA and bithiazole ring of Bm. Bm and BLMA are indicated in green and red, respectively. Phe33 and Phe38 are located on the BLMA surface at the bottom of the long groove. (b) The complex structure of BLMA and Bm. Red, blue, yellow and purple indicate acidic, basic, aromatic (Pro, Phe, Trp, His) and the other residues, respectively. The green color indicates the energy minimized Bm molecule. The metal-binding site and the terminal amino moiety of the Bm molecule are located upper and lower, respectively. Structures encircled in black are ¯exible. Residues 84-92 and 99-103 form large loop regions. This Figure was prepared using the program Discover on Insight II (Biosym Co., Ltd.). orthorhombic system. The space group was P21212, and Ê , b ˆ 67.94 A Ê and the cell dimensions were a ˆ 54.90 A Ê (Kumagai et al., 1998). c ˆ 35.60 A Weissenberg photographs of the native and derivative crystals were taken at BL-6B in the Photon Factory, the National Laboratory for High Energy Physics, Tsukuba, Japan. This beamline was constructed for the Tsukuba Advanced Research Alliance (TARA) project of Tsukuba

University (Sakabe et al., 1995). In this beamline, the crystal-to-®lm distance was 547 mm and the detectors were a pair of 800 mm  400 mm Imaging Plates (IPs) (Fuji Film Co., Ltd.) per photograph. Omega step scans were employed with a scan width of 25  . Each scan was 2  overlapped. The coupling constant was 1.5  /mm. Only four photographs were taken for one complete data collection from one crystal. The diffraction data of

Table 1. Statistics of data collection Crystal

Conditions for derivatives

Ê) Resolution (A

Total reflections

Unique reflections

Completeness (%)

Rmerge

Native PCMBS

20 mM, 24 h

1.5 2.0

74,090 35,870

19,723 9236

94.3 97.9

0.054 0.039

Rmerge ˆ h,jjIhj ÿ hIhij/hhIhi, where hIhi is the mean intensity of re¯ection h and Ihj is the jth measurement of re¯ection h.

923

Crystal Structure of a Bleomycin-binding Protein Table 2. Phasing statistics FOM

Acentric

0.5697

22.9

Diso

Centric

Dano all

35.0

8.9

Phasing power Acentric Centric 2.47

1.89

Acentric

Rcullis Centric

Anomalous

0.54

0.49

0.85

FOM ˆ h(jF(best)jjFj)i; Diso ˆ hkljFPH ÿ FPj; Dano ˆ hkljF(‡) ÿ F(ÿ)j; phasing power ˆ [(hklFH(calc)2/hkl(FPH(obs)ÿ FPH(calc))2]1/2; Rcullis ˆ hkljjFPH  FPj ÿ FH(calc)j/hkljFPHjFPH  FPj; F(best) is the calculated structure factor from the best phase angle. FPH, FP and FH are the derivative, native and heavy-atom structure factor amplitudes, respectively. F(‡) and F(ÿ) are heavyatom structure factor amplitudes of Friedel pairs.

Ê the native crystal were obtained at a resolution of 1.5 A Ê wavelength). Data using monochromatic X-rays (1.0 A processing was carried out with the program WEIS (Higashi, 1989) and the CCP4 (1994) program suite. The merging R-value was 5.4 % for the native crystal at a Ê (Table 1). resolution of 1.5 A

were re®ned and initial phases for the native protein crystal were calculated from the re®ned mercury atom position using the single isomorphous replacement method including anomalous scattering effect (SIR-AS) using CCP4 program mlphare. Phasing statistics are shown in Table 2.

Heavy-atom derivative search and phase determination

Model building and structure refinement

Heavy-atom derivatives were made by soaking crystals in mother liquor containing various concentrations of heavy-atom compounds. Soaking times were for 24 hours or longer. The soaked crystals were used for Ê . Each data set diffraction data collections up to 2.0 A was scaled to the native data set. Difference Patterson maps were calculated to search the heavy-atom binding sites using the CCP4 program. After six trials of heavyatom compounds, only the p-chloromercuribenzene sulfonic acid (PCMBS) derivative generated clear peaks in its difference Patterson maps. The position of the heavy-atom was clearly determined from the Harker sections of the isomorphous difference Patterson map (x ˆ ÿ 0.1339, y ˆ ÿ 0.1597, z ˆ ÿ 0.1750), and this peak was also located in the anomalous difference Patterson map. The position and occupancy of the mercury atom

To improve the quality of the electron density map, a density modi®cation was carried out by solvent ¯attening using the program dm (Wang, 1985). The solvent Ê. level was set to 45 % and resolution range was 20-2.0 A The high-quality electron density map allowed the tracing of the main-chain atoms for almost of all residues, except those from Asp85 to Ser90, Pro101, Arg109 and the C terminus. Side-chain atoms were built into the sequence for most residues on the initial electron density map, except Lys3, Glu30, Lys31, Arg42, Ile46, Arg47, Arg52, Glu54, Glu67, Glu76, Glu77, Arg80, Val82, Glu99 and Glu105. The model building was carried out with the program TURBO-FRODO (Jones, 1978) on a Silicon Graphics work station. The structure re®nement was Ê resolution using started with the data set of 20-2.0 A program X-PLOR (BruÈnger, 1990). Ten percent of the total re¯ections was excluded for free R-factor (Rfree)

Ê ; threshold Figure 11. Final 2Fo ÿ Fc electron density map and structure of the BLMA molecule. Resolution 10-1.5 A level 2s.

924

Crystal Structure of a Bleomycin-binding Protein

Table 3. Results of structure re®nements Resolution range of refinement Ê) (A Number of protein atoms Number of water molecules Sigma cut off Intensity R.M.S. deviation Ê) Bond length (A Bond angles (deg.) Impropers (deg.) Rwork (%) Rfree (%)

10.0-1.5 934 91 jFjs > 2.0 0.001 < jFj < 1000000 0.005 1.24 0.65 19.0 22.1

Rwork ˆ hkljFobs ÿ Fcalcj/hklFobs, where Fobs and Fcalc are the observed and calculated structure factor amplitudes, respectively. Rfree, 10 % of the complete data set was used for calculation.

calculations (BruÈnger, 1992) to monitor the re®nement progress. The initial value of the conventional R-factor (Rwork) was 44.3 % and after the simulated annealing, Rwork was quickly reduced to 27.7 %. All main-chain atoms and most side-chain atoms, except residues Glu30, Glu54, and Arg80, were clearly de®ned using an omitmap (Fo ÿ Fc map with calculated structure factor amplitudes and phases from the omitted structure). The resolÊ to 1.5 A Ê. A ution was extended stepwise from 2.0 A single molecule of BLMA was inspected and adjusted using the difference Fourier maps and the omit maps. If an unidenti®ed peak appeared in the difference Fourier Ê distance from the nitrogen or oxymap, with a 3.5-2.0 A gen atoms of BLMA, the peak could be assigned to a water molecule. Alternative conformations for sidechains of residues Lys31, Arg36, Ile66, Glu105 and Thr119 were adjusted using the difference Fourier map and the omit maps. Occupancies were restricted for all side-chain atoms of each alternative conformation. Water molecules were rejected when the B-factors were greater Ê 2 or corresponding peaks were not found in than 60 A the difference Fourier map. The Rwork and Rfree values for ®nal coordinates including 91 water molecules were 19.0 % and 22.1 %, respectively. A part of the 2Fo ÿ Fc electron density map calculated using the ®nal coordinates is given in Figure 11. Deviations from ideal geometry for the ®nal structure are shown in Table 3. The Ramachandran plot (Ramachandran & Sasisekharan, 1968) produced by PROCHEK (Laskowski et al., 1993) shows that all non-glycine residues fall within the allowed region and 94.2 % of all residues fall within the most favored regions. According to the method of Luzzati (1952), the mean error of the coordinates was Ê. estimated to be about 0.2 A

Measurement of a half-saturating concentration (K1/2) for BLMA/Bm complex Bm A2 sulfate (at a range of 0.05 to 0.5 mM) was added to 2 ml of 1 mM Tris-HCl buffer (pH 7.5) containing 0.4 mM BLMA. Experiments were carried out with a spectro¯uorophotometer (model RF-5000, Simadz, Japan); excitation 245 nm, emission 330 nm, slit-width at excitation 10 nm, and slit-width at emission 10 nm. The present study used molar absorptivity e ˆ 20,265, calculated from the number of Trp and Tyr residues in the BLMA molecule (13,292 Da), to determine the concentration of protein dissolved in solution.

BLMA can use to its advantage the strong ¯uorescence emission, with a maximum at 330 nm, when the protein is excited at 245 nm in aqueous solution, due to three tryptophan residues in the molecule. The protein's ¯uorescence in solution is progressively quenched by addition of increasing concentrations of Bm even after correction of the inner ®lter effect. When the reciprocal of the measured decrease of the ¯uorescence intensity is plotted against the reciprocal of added Bm concentration, a straight line is obtained, with a regression coef®cient greater than 0.995, from which a half-saturating concentration (K1/2) is calculated. Brookhaven Protein Data Bank accession number The coordinates have been deposited in the Brookhaven Protein Data Bank, accession number 1QTO.

Acknowledgments This work was supported, in part, by the Ministry of Education, Science, Sports and Culture, Japan, and the President's Special Research Grant of RIKEN by the SR Structural Biology Research Group of RIKEN, Japan. We are grateful to Dr N. Kamiya of RIKEN Harima Institute for his valuable discussion about X-ray protein crystallographic analysis. We thank Dr N. Sakabe of Tsukuba University and Drs N. Watanabe and M. Suzuki of the National Laboratory for High Energy Physics, for their kind help in data collection at the Photon Factory. We thank Mr N. Kimura of CTC Laboratory Co., for his kind support in the use of the program Insight II. We are grateful to Mr T. Miyazuki, Nippon Kayaku Co., Ltd., for the gift of bleomycin A2 sulfate.

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Edited by I. A. Wilson (Received 29 April 1999; received in revised form 8 November 1999; accepted 18 November 1999)