The solution structure of a cytotoxic ribonuclease from the oocytes of Rana catesbeiana (bullfrog)1

Article No. mb982082

J. Mol. Biol. (1998) 283, 231±244

The Solution Structure of a Cytotoxic Ribonuclease from the Oocytes of Rana catesbeiana (Bullfrog) Chi-Fon Chang, Chinpan Chen, Yi-Cheng Chen, Kellie Hom Rong-Fong Huang and Tai-huang Huang* Division of Structural Biology Institute of Biomedical Sciences Academia Sinica, Nankang Taipei, 11529, Taiwan The Republic of China

RC-RNase is a pyrimidine-guanine sequence-speci®c ribonuclease and a lectin possessing potent cell cytotoxicity. It was isolated from the oocytes of Rana catesbeiana (bull frog). From analysis of an extensive set of 1H homonuclear 2D NMR spectra we have completed the resonance assignments. Determination of the three-dimensional structure was carried out with the program X-PLOR using a total of 951 restraints including 814 NMR-derived distances, 61 torsion angles, and 76 hydrogen bond restraints. In the resultant family of 15 best structures, selected from a total of 150 calculated structures, the root-mean-square deviation from the average structure for the backbone heavy-atoms involved in Ê , while that for all backbone well-de®ned secondary structure is 0.48 A Ê heavy-atoms is 0.91 A. The structure of RC-RNase consists of three a-helices and two triple-stranded anti-parallel b-sheets and folds in a kidney-shape, very similar to the X-ray crystal structure of a homologous protein, onconase isolated from Rana pipiens. We have also investigated the interaction between RC-RNase and two inhibitors, cytidylyl(20 ! 50 )guanosine (20 ,50 -CpG) and 20 -deoxycytidylyl(30 ! 50 )-20 deoxyguanosine (30 ,50 -dCpdG). Based on the ligand-induced chemical shift changes in RC-RNase and the NOE cross-peaks between RC-RNase and the inhibitors, the key residues involved in protein-inhibitor interaction have been identi®ed. The inhibitors were found to bind in a ``retro-binding'' mode, with the guanine base bonded to the B1 subsite. The His103 residue was found to occupy the B state with the imidazole ring pointing away from the active site. The structure coordinates and the NMR restraints have been deposited in the Brookhaven Protein Data Bank (1bc4 and 1bc4mr, respectively). # 1998 Academic Press

*Corresponding author

Keywords: RC-RNase; cytotoxic protein; sialic acid; NMR; lectin

Present address: K. Hom, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446, USA Abbreviations used: RC-RNase, ribonuclease from the oocyte of Rana catesbeiana; 20 ,50 -CpG, cytidylyl(20 !50 )guanosine; 30 ,50 -dCpdG, 20 deoxycytidylyl(30 !50 )-20 -deoxyguanosine; sialic acid, N-acetylneuraminic acid; 60 -sialyllactose, 60 -N-acetylneuramin-lactose; DSS, (2,2-dimethyl-2silapentane-5-sulfonate); RMSD, root-mean-square deviation; 2D, two-dimensional; 3D, three-dimensional; ppm, parts per million; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; DQF-COSY, double quantum ®ltered correlated spectroscopy; TOCSY, total correlated spectroscopy; FID, free induction decay; TPPI, time-proportioned phase sensitive incrementation. E-mail address of the corresponding author: [email protected] 0022±2836/98/410231±14 $30.00/0

Introduction RC-RNase is a pyrimidine-guanine sequencespeci®c ribonuclease isolated from Rana catesbeiana (bullfrog) oocytes (Liao, 1992). The primary sequence of RC-RNase is identical with that of a sialic-acid-binding lectin (SBL-C) isolated from the oocytes of R. catesbeiana (Nitta et al., 1987; Titani et al., 1987; Okebe et al., 1991; Chen et al., 1996) and is highly homologous to that of an RNase isolated from the liver of R. catesbeiana (Titani et al., 1987; Nitta et al., 1989). It is also relatively similar to those of human angiogenin (Kurachi et al., 1985), onconase isolated from Rana pipiens eggs (Mikulski et al., 1990; Ardelt et al., 1991), and bovine pancreatic RNase A (Beintema et al., 1984). Thus, this protein belongs # 1998 Academic Press

232 to the superfamily of pyrimidine base-speci®c RNases (Nitta et al., 1993, 1994). In addition to possessing ribonucleolytic activity, RC-RNase is cell cytotoxic, similar to onconase protein (Sakakibara et al., 1979; Ardelt, et al., 1991, 1994; Wu et al., 1993, 1995; Liao et al., 1996; Youles & D'Alessio, 1997). It inhibits the growth of tumor cells such as P388 and L1210 leukemia cells in vitro and is effective for in vivo killing of Sarcoma 180, Ehrlich, and Mep II ascites cells (Nitta et al., 1994). The activity of RC-RNase in the oocyte has been found to be regulated by both inhibitor binding and compartmentation (Liao & Wang, 1994), with 99% of the protein found in the yolk granules and the remaining 1% in the cytosol (Liao et al., 1996). In contrast, no cytotoxicity or lectin activity was found for either bovine pancreatic RNase A or angiogenin. The unique cytotoxic and antitumor activities of onconase and RC-RNase are now being exploited for their medical applications. Onconase is currently undergoing phase III clinical test as an antitumor drug and the potential of RC-RNase as an anticancer drug is also under evaluation (Youles & D'Alessio, 1997). The three-dimensional structures of several RNase A/inhibitor complexes have been solved to high resolution by X-ray crystallographic and NMR methods (for reviews, see Kolbanovskaya et al., 1993, D'Alessio & Riordan, 1997). Similarly, the structures of several RC-RNase homologues, including angiogenin (Acharya et al., 1994, 1995; Lequin et al., 1996), onconase (Mosimann et al., 1994), porcine pancreatic procolipase (Breg et al., 1995), and guanine-speci®c ribonuclease F1 (Nakai et al., 1992) have been determined. The putative active-site residues of these proteins have been identi®ed. Several binding subsites for various parts of the nucleotide substrate have been proposed and the main residues involved in substrate binding for some of these subsites have been implicated (Richards et al., 1971; Pares et al., 1991; Zegers et al., 1994; Toiron et al., 1996). An interesting alternative ``retro-binding'' mode has been found in high-resolution X-ray crystal structures of RNase A soaked in 20 ,50 -CpG and 30 ,50 -dCpdG solutions (Aguilar et al., 1989, 1991, 1992). In this novel, non-productive binding mode, the guanine base (B2 in conventional designation) binds to the pyrimidine B1 binding site adjacent to Thr45, while the traditional B2 purine binding site is unoccupied. A solvent SO2ÿ 4 anion occupies the P1 site and is not displaced by the phosphate group of the inhibitor molecule. Instead, the CMP or dCMP moiety of the inhibitor molecule is held loosely in a channel running towards the surface of the protein molecule, and is thus completely external to the active site. it has been shown that this mode of binding is not an artifact, but is indeed a phenomenon speci®c to mono- or dinucleotides containing guanine (Mills et al., 1992). However, this ``retro-binding'' mode has not been shown to exist in solution.

Solution Structure of a Cytotoxic Ribonuclease

At present, the three-dimensional structure of RC-RNase has not been reported and the structural basis of the functional differences among these proteins is not fully understood. Of particular interest is the location of the lectin-binding site. We have undertaken the task of determining the solution structure of RC-RNase by NMR methods. The proton NMR resonance assignments and the secondary structures deduced from NMR data have been published (Chen et al., 1996). In comparison, RC-RNase (111 residues) and onconase (104 residues) are smaller than RNase A (124 residues) and angiogenin (125 residues); only angiogenin has three disul®de bonds, while there are four in the other three ribonucleases. The common features in the structures of these proteins are two anti-parallel b-sheets, arranged in a bowl shape, and three a-helices, located at similar positions. Both RNase A (Rico et al., 1991; Santoro et al., 1993; Toiron et al., 1996) and angiogenin (Reisdorf et al., 1994) have seven b-strands, compared to six observed in onconase (Mosimann et al., 1994) and RC-RNase. Our NMR data indicated that the a-2 helix in RC-RNase is less stable than the corresponding helices in the other RNases. Here, we present the solution structure of RC-RNase calculated from NMR constraints. We report our observation on the interaction of RC-RNase with two inhibitors, cytidylyl(20 ! 50 )guanosine (20 ,50 CpG) and 20 -deoxycytidylyl(30 ! 50 )-20 -deoxyguanosine (30 ,50 -dCpdG). Our results con®rm the retrobinding mode reported in the high-resolution X-ray crystal structures of RNase A/20 ,50 -CpG and RNase A/30 ,50 -dCpdG complexes.

Results and Discussion Complete backbone 1H resonance assignments Since RC-RNase was directly puri®ed from bullfrog oocytes, we could not apply heteronuclear multi-dimensional NMR to aid our resonance assignments. Nevertheless, from analyzing a large set of two-dimensional proton NMR spectra we have assigned the backbone and side-chain proton resonances of all but ®ve residues (Chen et al., 1996). Four of these ®ve unassigned residues, with the exception of the N-terminal pyroglutamate residue, were identi®ed (Table 1) during the course of the structure determination. We observed an intense NOE cross-peak between the two Ca protons of residues Arg79 and Pro80 (data not shown), indicating that the peptide bond of Pro80 is predominantly in the cis conformation. The conformations of the other four proline residues (Pro15, Pro78, Pro82 and Pro111) were identi®ed as trans, due to the observation of large NOE cross-peaks between the CdH and the CaH groups of the preceding residue for each of these proline residues. The completed assignments have been deposited in the BMRB data bank (BRMB 4138).

233

Solution Structure of a Cytotoxic Ribonuclease Table 1. Additional 1H NMR chemical shifts of RC-RNase (pH 3.5, 310 K) 1

Residue

CaH

Pro78 Arg79 Pro80 Pro111

4.49 4.18 4.68 4.70

CbH 1.77, 1.90, 2.03, 1.93,

H NMR chemical shifts (ppm) CgH

1.65 1.84 2.03 1.93

1.96, 1.69, 2.22, 2.24,

1.89 1.69 2.22 2.24

Others

CdH2 CdH2 CdH2 CdH2

3.48, 3.17, 3.62, 3.54,

3.67 3.17 3.50 3.47

This Table completes the assignment previously determined (Chen et al., 1996). The Chemical shifts are referred to DSS calibrated at 0.00 ppm.

Structural statistics There are 1381 restraints obtained, including 628 intra-residue, 269 sequential, 347 medium and long-range inter-proton distances, 76 hydrogen bonds, 46 f torsion angles, and 15 w1 torsion angles. In the structure calculation, we used only 951 restraints, excluding 430 intra-residue NOE restraints. Fifteen stereospeci®c assignments have been made, which allow us to set more precise distance restraints for over 80 NOE cross-peaks (Figure 1). Figure 2(a) shows the number of restraints per residues employed for the calculation. A total of 150 structures was calculated using the program X-PLOR 3.1 (BruÈnger, 1992). From these structures, 15 with the lowest target function and the minimal distance and torsion angle restraint violations were chosen for evaluation. Table 2 summarizes these structural statistics. These 15 structures are all consistent with both experimental data and standard covalent geometry. Analysis of the 15 structures with the

Figure 1. Overlay of a selected aliphatic region of the DQF-COSY (red) and TOCSY (black) spectra, obtained at 310 K with a 2 mM protein sample in 50 mM phosphate buffer in 99.8% 2H2O, pH 3.5. The 15 stereospeci®cally assigned residues are annotated and labeled b1 and b2 for the methylene protons according to the convention of X-PLOR-3.1

mean structure yields an average root-mean-square Ê for all backbone deviations (RMSD) of 0.91 A

Figure 2. Bar diagrams showing the structural statistics of the solution structure of RC-RNase. (a) Number of NOE distance restraints for each residue. White, intra-residue NOE restraints; gray, sequential NOE restraints; dark gray, inter-residue NOE restraints. (b) Root-mean-square deviation (RMSD) of the backbone atoms. (c) Root-mean-square deviation (RMSD) of the side-chain atoms. RMSDs are determined by calculating the root-mean-square deviation of the backbone or sidechain positions of the 15 best structures with respect to the positions of the average structure and are calculated with X-PLOR program.

234

Solution Structure of a Cytotoxic Ribonuclease

Table 2. Summary of the statistics for the 15 converged structure of the RC-RNase Restraint totals by type

Number

Intra-residue restraints (i, j, i ˆ j) Sequential restraints (i, j, j ˆ i ‡ 1) Inter-residue restraints (i, j, i ‡ 1 < j) Hydrogen bond restraints Torsion angle restraints Total restraints XPLOR energies (kcal molÿ1) Ebond Eangle Eimproper Evdw Emoe Ecdih Etotal RMSD from ideal geometry Ê) Bonds (A Angles (deg.) Impropers (deg.) RMSD from experimental restraints Distance restraints Torsional restraints Ê) Atomic RMSD (N, Ca, C0 , O) (A hSAi versus hSAisec.struct hSAi versus hSAiall residues

198 269 347 76 61 951 hSAi 84.003 437.362 87.014 243.230 291.208 28.507 1171.323 hSAi 0.007 0.954 0.793 hSAi 0.128 9.748 0.48 0.91

Notation is as follows: hSAi is the ensemble of 15 ®nal X-PLOR structures. hSAisect.struct is the average coordinates for residues involved in secondary structure that were obtained from a least-squares superposition of the backbone (N, Ca, C0 and O) heavy atoms. hSAiall residues is the average coordinates for all residues (1 to 111) from a least-squares superimposition of all backbone heavy atoms.

Ê for backbone atoms in the heavy-atoms and 0.48 A well-de®ned secondary structure regions (Trp3 to Ile11, Asn18 to Met23, Val37 to Ile42, Ala45 to Thr53, Asn57 to Ser62, Phe66 to Arg73, Tyr83 to Asn90, Ile92 to Glu97 and Tyr100 to Gly106). Figure 2(b) and (c) show the RMSD for backbone and side-chain of each residue, respectively. The least-de®ned regions are the C terminus (residues 106 to 111) and the loop region between residues Ser75 and Cys81. The loop regions Ile13 to Asn18 and Asp24 to Arg36 are also less de®ned, due to fewer NOE distance restraints detected. The RMSD for helix a2, Asn18 to Met23, is considerably larger than that of the other helical regions, consistent with the observation of faster amide proton exchange rates for residues in this region. The N terminus is better de®ned, presumably due to the formation of helix a1 from Trp3 to Ile11. The quality of the structure is good, as judged by the low Ê ), bond RMSD values for bond lengths (0.007 A angles (0.954 ) and impropers (0.793 ) from the ideal geometry. Furthermore, most of the backbone torsion angles for non-glycine and non-proline residues (97.4%) fall in the allowed regions and almost all (99.8%) of them fall in the generally allowed regions (Figure 3). Structure description The superimposition of the 15 calculated best structures is shown in Figure 4. In general, the

Figure 3. Ramachandran (f, c) plot for the 15 structures of RC-RNase. The degree of preference regions in the Ramachandran plot are represented by different colors. Red, the most favorable; bright yellow, allowed; light yellow, generally allowed; and white, disallowed. Glycine residues are represented by triangles. This plot was generated with the PROCHECK-NMR program (Laskowski et al., 1993).

conformation is well de®ned, with the exceptions of the loop and C terminus regions, as noted above. Figure 5(a) and (b) shows two views of the RIBBONS (Carson, 1987, 1991) representations of the energy-minimized average structure of RC-RNase. The secondary structure of RC-RNase consists of six b-strands and three a-helices. The dominant feature of the tertiary folding is the presence of two twisted, triple-stranded anti-parallel b-sheets forming a bowl shape, with the planes of the two sheets at about 120 apart. The overall dimension (backbone to backbone) of the protein is Ê  28 A Ê  28 A Ê . One of the anti-parallel about 46 A b-sheets (S1, strands b1, b3 and b4) is well de®ned while the other (S2, strands b2, b5 and b6) is more disordered and highly distorted. The NOE pattern, amide proton exchange rates, and the 3JNHa for residues in the three regions, Val30 to Gln33, Thr53 to Ile56, and Glu97 to Tyr100 are consistent with these residues being in b-turn structures. Furthermore, the observation of daN(97,99), daN(97,100), dNN(97,100) suggests that residues Glu97 to Tyr100 form a type I b-turn structure. Long-range NOEs between this turn and residues Ile56 and Met58 were observed. Tyr91, which connects b4 and b5, appears to form a b-bulge. The well-de®ned N-terminal a1 helix is located between the two b-sheets and is oriented approximately perpendicular to strand b1. The other two helices, one on each side of helix a1, are located on the same side of the twisted plane formed by the two sheets and with

235

Solution Structure of a Cytotoxic Ribonuclease

Figure 4. Stereo view of the superimposition of the backbone of 15 best NMR solution structures selected from 150 structures calculated with 951 NMR constraints.

their axes oriented approximately perpendicular to the plane. Helix a2 is packed at the outer edge of b4 and is attached to S1 through the Cys19-Cys71 disul®de bond linkage between the C-terminal end of helix a2 and the b3 strand (Figure 6, colored magenta). Helix a2 is oriented approximately parallel with a1. Helix a3 is packed against the second b-sheet, S2, through a disul®de bond Cys52-Cys96 between the C-terminal of a3 and b5 in S2. It is oriented perpendicular to the other two helices. The third disul®de bond, formed between Cys34 and Cys81, joins together the two loops on the S1 side, while the fourth disul®de bond, Cys93Cys110, anchors the C terminus to the b5 strand of S2 sheet.

Figure 5. Ribbon diagram of (a) and (b) RC-RNase, (c), onconase and (d), bovine pancreatic RNase A generated with the program RIBBONS (Carson, 1987, 1991). The coordinates for the structure of RC-RNase are from the energy minimized best structure of the 15 best structures shown in Figure 4. The coordinates of onconase (Mosimann et al., 1994, 1ONC) and RNase A (Santoro et al., 1993, 2AAS) were taken from Brookhaven Protein Data Bank.

Structural heterogeneity In the course of NMR resonance assignment, we observed two amide proton resonances for each of the following 11 residues; Ile17, Ile22, Val37, Ile42, Phe66, Gln67, Leu68, Asn69, Arg86, Thr87 and Glu88 (Chen et al., 1996). We observe two a-protons for each of the following eight residues; Val37, Ile42, Phe66, Gln67, Leu68, Asn69, Thr87 and Glu88. Two sets of resonances were observed for the side-chain protons in several of these residues. Mass spectroscopy and gel electrophoresis analysis showed that this protein is over 95% pure (Chen et al., 1996). The locations of these residues are shown in green in Figure 6. Nine of these residues are located in the b1, b3 and b4 strands, which cover a large portion of the S1 sheet. The remaining two residues are located in loop 1 and the a2 helix, and are spatially not far from the S1 sheet. Thus, this region appears to exist in two conformations. From the NOE cross-peak intensities, we estimated the population of these two conformers to be 40:60 at 320 K. Since many of these residues are located in the binding pocket we suspect that the biological activities of these two conformers

Figure 6. A ribbon representation of the best structure of RC-RNase, showing the locations of the disul®de bonds (magenta), the proline residues (red) and the residues that exist in two conformations (green).

236 may be quite different. Interestingly, Tyr83, which showed only one resonance each for the amide and a protons, displayed four ring proton resonances in contrast to the two averaged ring proton resonances generally observed for tyrosine residues in single conformation yet undergoing fast ring ¯ipping motion; in addition, six cross-peaks among these protons were observed in both TOCSY and NOESY spectra at 310 K. Lowering the temperature to 280 K resulted in the observation of only two cross-peaks. On the other hand, at 320 K the cross-peaks broadened substantially. When the temperature was raised to 335 K, all the cross-peaks disappeared, presumably due to excessive exchange broadening. We interpreted this observation as due to the presence of exchange of two conformers for Tyr83. Inspection of the RC-RNase structure showed that the Tyr83 ring is located in a loop consisting of three proline residues (Pro78, Pro80 and Pro82; Figure 6, colored red) near the most heterogeneous region of the structure and is likely to exist in two magnetically different environment, thus, supporting our speculation (Chen et al., 1996). However, we cannot rule out the possibility that the exchange behavior of Tyr83 ring protons are due to ring ¯ipping, which was observed quite often. Similarly, in the highresolution phosphate/sulfate-free RNase A crystal structure, 13 residues are found to exit in two conformations (Glu11, Ser32, Asn34, Val43, Ser50, Lys61, Asn67, Ser77, Asp83, Arg85, Lys91, Lys98 and Lys104; Svensson et al., 1986; Wlodawer et al., 1988). In comparison, the structural heterogeneity in RNase A is much more pervasive, with the residues exhibiting multiple conformation scattered all over the molecule. In RC-RNase, however, residues displaying two conformations are more localized. It is possible that conformational heterogeneity of only a few residues in key positions may be responsible for a concerted structural change in the S1 sheet in RC-RNase. More extensive studies are needed to further characterize the origin of structural heterogeneity and the functional signi®cance of the two conformers. Comparison with other ribonucleases At present, the three-dimensional structures of several RC-RNase homologues, with and without added inhibitors, have been determined by NMR or X-ray crystallographic methods (for a review, see D'Alessio & Riordan, 1997). In this section we compare the structure of RC-RNase with the structures of two most relevant proteins, i.e. RNase A and onconase. Overall folding RC-RNase belongs to the pancreatic ribonuclease superfamily. It shares 27% sequence identity with the mammalian bovine pancreatic RNase A and 53% sequence identity with the amphibian onconase. However, in addition to the

Solution Structure of a Cytotoxic Ribonuclease

ribonucleolic activity, both RC-RNase and onconase have been shown to be lectins, and both are cytotoxic with unique antitumor activities. In contrast, no cytotoxicity or lectin activity was found for bovine pancreatic RNase A. The secondary structure features of these proteins have been compared (Chen et al., 1996). The present result further reveals the structure similarity among these proteins. In spite of the differences in polypeptide chain length (124, 111 and 104 amino acid residues for bovine pancreatic RNase A, RC-RNase and onconase, respectively), these three proteins all possess two anti-parallel b-sheets and three a-helices, folded in the bowl (kidney) shape (Figure 5). The location of the three helices is similar in all three ribonucleases. The similarity in tertiary folding of the three proteins is manifested in the three long-range disul®de bonds (Figure 6, colored magenta). The ®rst disul®de bond between Cys26 and Cys84 (RNase A sequence, Cys19-Cys71 in RC-RNase) anchors the second helix a2 to the S1 sheet through its attachment to the b4 (b3 in RC-RNase and onconase) strand. The second disul®de bond, Cys40Cys95 (Cys34-Cys81 in RC-RNase), joins together the two major loops on the far side of S1, thus ®xing the position of the a2 helix and the two loops. The third disul®de bond between Cys58 and Cys110 b (Cys52-Cys96 in RC-RNase) pulls the third helix toward the b6 (b5 in RC-RNase) strand. The fourth disul®de bond in RNAse A, Cys65-Cys72, is more localized. It connects b2 and b3 in such a way as to cause the twist in the S1 sheet by forcing b2 and b3 to orient in a some what odd position. In the case of RC-RNase, this disul®de bond is in the C terminus (Cys93Cys110), thereby limiting the ¯exibility of the C terminus. This type of disul®de bonding pattern causes the three proteins to adopt the bilobate, kidney-shaped structure. A more careful inspection of the three structures does reveal some differences. In particular, the higher degree of sequence homology between RCRNase and onconase is translated into higher degree of structure homology between these proteins. Larger differences occur primarily in the S2 region, where four b-strands are present in RNase A and only three are present in both RCRNase and onconase. The relative orientation of the three helices appears to be different in the three proteins. Despite these differences, the common prominent feature of these proteins is the presence of a central crevice. As shown in the GRASP (Nicholls et al., 1991) representation of RC-RNase (Figure 7), this crevice is also the most positively charged region, with several lysine residues scattered around. This pocket has been identi®ed as the active-site region in both RNase A and onconase. Our inhibitor-binding studies described below con®rmed that the central crevice is also the substrate-binding site in RC-RNase.

Solution Structure of a Cytotoxic Ribonuclease

Figure 7. The surface structure and surface charge pro®le of the energy minimized, averaged structure of RC-RNase. The structure was generated with the program GRASP (Nicholls et al., 1991) with partial charge taken directly from the default charge table (full.crg). Blue represents positive electrostatic potential, red represents negative electrostatic potential and white indicates charge neutral regions.

Active-site geometry The important residues involved in catalytic activities of RNase A (Gln11, His12, Lys41, Thr45, His119 and Phe120) have been identi®ed. The counterparts in onconase are Lys9, His10, Lys31, Thr35, His97 and Phe98 (Mosimann et al., 1994). In RC-RNase, they are Lys9, His10, Lys35, Thr39, His103 and Phe104. The GRASP (Nicholls et al., 1991) representation of the surface structure of RC-RNase shown in Figure 7 clearly displays the presence of a deep binding pocket enclosed by S1 and S2 on the two sides and capped on one end by the a1 helix. The putative catalytic residues of

237 RC-RNase are all clustered within this pocket: Residues Lys9 and His10 are on helix a1; Lys35 and Thr39 are on the b1-strand; His103 and Phe104 are on the b6-strand. Such an arrangement can certainly accommodate the extended binding of ligands, as was observed by Fontecilla-Camps et al (1994). Interestingly, two negatively charged glutamate residues (Glu97 and Glu88) cap the two ends of the groove, seemingly positioned to repel the negatively charged phosphate group of RNA. The spatial arrangement of the some active-site residues for the three proteins are shown in Figure 8. The relative location of these active-site residues among the three proteins are remarkably similar. One major difference is the relative orientation of the two important catalytic histidine residues. In RNase A (colored yellow), His119 has been found to exist in two possible conformations, A and B (Borkakoti et al., 1982; Rico et al., 1991, 1993; Santoro et al., 1993; Zegers et al., 1994). In conformation A, corresponding to the active conformation, His119 points inward toward the center of the active site. In the inactive conformation B (not shown), the imidazole ring of His119 points outward. Inspection of the conformations of the onconase (colored magenta) and RC-RNase (colored green for all 15 best structures) in Figure 8 showed that the corresponding histidine (His97 in onconase and His103 in RC-RNase) residues in these two proteins occupy the B conformation, with their imidazole rings pointing away from the pocket. The RMSDs of the side-chain of these Ê , 1.9 A Ê, active-site residues in RC-RNase are 1.6 A Ê , 0.5 A Ê and 0.5 A Ê for Pyr1, His10, Lys35, 1.5 A Thr39 and His103, respectively. Thus, Pyr1, His10 and Lys35 are not well de®ned, whilst Thr39 and His103 are well ordered. Preliminary 15N relaxation data for onconase showed that the order parameters of the two catalytic histidine residues are small, suggesting the presence of dynamics ¯uctuation of the orientations of these two residues

Figure 8. Spatial arrangement of the active-site residues of RC-RNase (green), onconase (magenta) and RNase A (yellow). Residues are labeled according to the RC-RNase numbering scheme, except Lys1, which is the ®rst residue of bovine pancreatic RNase A. The ribbon was that of the RC-RNase structure with least restraint violations. Superimposition of the 15 selected structures of RC-RNase are shown to show the structural disorder of the side-chains of Ê , 1.9 A Ê , 1.5 A Ê , 0.5Ê and 0.5 A Ê for Pyr1, His10, Lys35, Thr39 these residues. RMSD values of these residues are: 1.6 A and His103, respectively.

238

Solution Structure of a Cytotoxic Ribonuclease

(unpublished results). Some of these differences probably account for the lower ribonucleolytic activities of onconase and RC-RNase, compared to that of RNase A. It should be pointed out that, since the binding pocket is solvent-exposed, these active-sites residues are on the surface of the protein and are expected to be ¯exible. Thus, their orientations can change considerably upon ligand binding.

used; therefore, the presence of hydrogen bonds between Pyr1 and the a1 helix or the C-terminal b-sheet, if any, could not be con®rmed in the present study. Furthermore, from the proton spectrum of the unlabeled RC-RNase protein, we can assign only the CaH proton of Pyr1 , and the conformation of Pyr1 is rather unde®ned. Recently, we have prepared 15N and 13C uniformly labeled onconase samples and we are well on our way in determining its solution structure (unpublished results). With heteronuclear triple resonance experiments, we expect to be able to de®ne the conformation of the Pyr1 at a higher resolution.

Structure of the N terminus pyroglutamate residue In RC-RNase and onconase the carboxyl groups of the N terminus glutamate residue form a cyclic bond with the terminal amino group and exists as pyroglutamate. The role of the N-terminal residue, pyroglutamyl residue (Pyr1), has been extensively studies for onconase (Boix et al., 1996). The replacement of this residue causes the decrease of cytotoxicity and enzyme activity, and thus a unique role of the pryoglutamyl residue in the active site of amphibian RNases is indicated. In RNase A (Figure 8, colored yellow), Lys1 extends far out of the active site and no speci®c role for this residue has been implicated. On the contrary, in onconase, Pyr1 folds back against the N-terminal a-helix and is hydrogen bonded to Val96 in the C-terminal b-sheet. Pyr1 also hydrogen bonds to Lys9, which simultaneously interacts with the main phosphate group of the substrate (Moismann et al., 1994). In the structure of RC-RNase, the Pyr1 residue occupies a position similar to that in onconase, suggesting that Pyr1 may also play important roles in cytotoxicity and catalysis of RC-RNase. However, we could not detect the amide proton of Pyr1 under all of the NMR conditions that we have

Inhibitor binding studies In order to identify the active-site residues responsible for ribonucleolytic activity, we have employed 2D NMR spectroscopy to investigate the interaction between RC-RNase and two inhibitors, 20 ,50 -CpG and 30 ,50 -dCpdG in 2H2O In all cases, addition of these two inhibitors causes shifts of speci®c resonances, suggesting that the inhibitors undergo fast exchange between free and bound states. The ligand-induced chemical shift changes in the proton resonances of the protein,
d, observed for the two inhibitors are listed in Table 3. The results showed that both inhibitors induce nearly identical chemical shift changes in the protein resonances and the changes are limited to the following eight residues; His10, Val37, Ile41, Asn69, Ile92, Cys93, Val94 and His103. The proton resonances of Val37 and His10 displayed the largest chemical shift changes. Figure 9 shows the structure of RC-RNase with spatial locations of the perturbed residues represented by van der Waals

Table 3. Chemical shift difference between RC-RNase free form and complexes in 2H2O at 305 K Residue

H10 V37

I41 N69 I92 C93 V94 H103

a

Atom

2H 4H aH bH gH gH NH NH NH aH aH bH bH NH aH aH bH 2H 4H

Free

20 ,50 -CpG complex

30 ,50 -dCpdG complex

dfreea

dcomplexb


dc

dcomplexb


dc

8.04 6.46 5.55 2.17 1.13 1.05 9.59 9.20 8.99 5.14 5.89 3.30 2.22 9.00 5.00 5.55 3.30 8.06 7.04

8.00 6.22 5.42 1.97 0.95 0.80 9.61 9.25 8.96 5.09 5.85 3.23 2.19 8.96 4.92 5.57 3.28 7.99 6.99

ÿ0.04 ÿ0.24 ÿ0.13 ÿ0.20 ÿ0.18 ÿ0.25 0.02 0.05 ÿ0.03 ÿ0.05 ÿ0.04 ÿ0.07 ÿ0.03 ÿ0.04 ÿ0.08 0.02 ÿ0.02 ÿ0.07 ÿ0.05

8.00 6.23 5.42 1.97 0.94 0.77 9.64 9.29 8.99 5.11 5.88 3.23 2.21 8.98 4.95 5.57 3.26 7.97 7.01

ÿ0.04 ÿ0.23 ÿ0.13 ÿ0.20 ÿ0.19 ÿ0.28 0.05 0.09 ÿ0.00 ÿ0.03 ÿ0.01 ÿ0.07 ÿ0.01 ÿ0.02 ÿ0.05 0.02 ÿ0.04 ÿ0.09 ÿ0.03

dfree is the chemical shift (ppm) for RC-RNase free form (pH* 7.56). dcomplex is the chemical shift (ppm) for RC-RNase/20 ,50 -CpG or RC-RNase/30 ,50 -dCpdG complex (pH*, 7.56), where inhibitor/protein ˆ 2:1. c
d ˆ dcomplex ÿ dfree. b

239

Solution Structure of a Cytotoxic Ribonuclease

Table 4. Intermolecular NOEs observed in complexes at 305 K, pH* 7.56 20 ,50 -CpG complex (inhibitor/protein 4;1) Guanosinea,b Residue Observed NOE (ppm) G(H10 ) G(H10 ) G(H10 ) G(H8) G(H8) G(H8) G(H8)

Figure 9. Spatial arrangement of the residues whose proton chemical shifts change upon binding of 20 ,50 CpG. These residues are labeled and are shown by van der Waals spheres.

spheres. The perturbed residues are all clustered around the active-site region identi®ed above. The overall structure of the protein, as elucidated by the cross-peak pattern in the NOESY spectra, were found to be quite similar in the free and in the complex forms, indicating that binding of the inhibitors did not cause large conformational changes, except in those regions where changes in chemical shifts were detected. Similar results were observed in inhibitor binding to RNase A (Santoro et al., 1993). On the other hand, nearly all proton resonances of the nucleotide inhibitors show detectable chemical shift changes (data not shown). Of the two nucleotides, proton resonances from 30 ,50 -dCpdG show much larger chemical shift changes, as large as 0.433 ppm for the H6 proton and 0.147 ppm for the H20 of the cytosine base are observed. The guanosine moiety appears to display smaller chemical shift changes. The binding of substrates and inhibitors to RNase A (or S) have been studied in detail. The RNA molecule binds to RNase A with a speci®c orientation with respective to the active site. The ribose, base, and phosphate groups on the 30 side of the cleavage site bind to the so-called R1, B1 and P1 sites, respectively (Richards & Wyckoff, 1973). These sites are located near the ®rst helix, which encompasses His12 and His119. The binding site of the phosphate group associated with the 50 hydroxyl group of the R1 ribose moiety is designated as the P0 subsite. The ribose, base and phosphate groups of the nucleotide that is on the other side of the cleavage site are associated with the R2, B2 and P2 subsites, respectively. The next base in the sequence is at the B3 subsite and so on. Kinetic and structural studies on RNase A lead to the identi®cation of the main residues in each subsites: B1, Thr45, Phe120 and Ser123; P1, Gln11, His12, Lys41 and His119; B2, Asn67, Gln69, Asn71 and Glu111; P2, Lys7 (Richards et al., 1971; Pares et al., `1991; Zegers et al., 1994; Toiron et al., 1996).

Val37 (CgH) Val37 (CgH) Thr70 (CgH) Val37 (CgH) Val37 (CgH) Val37 (CbH) Thr70 (CgH)

30 ,50 -dCpdG complex (inhibitor/protein 2:1) Val37 (CgH) G(H10 ) G(H10 ) Val37 (CgH) Val37 (CbH) G(H10 ) G(H10 ) Thr70 (CgH) G(H8) Val37 (CgH) G(H8) Val37 (CgH) G(H8) Val37 (CbH) G(H8) Thr70 (CgH)

0.65 0.86 1.20 0.65 0.86 1.86 1.20 0.74 0.93 1.94 1.19 0.74 0.93 1.94 1.19

a For the 20 ,50 -CpG complex, chemical shift of G(H10 ) 6.00 ppm, G(H8) 8.15 ppm. b For the 30 ,50 -dCpdG complex, chemical shift of G(H10 ) 5.98 ppm, G(H8) 8.13 ppm.

Many of the corresponding residues in RC-RNase, such as Gln8, His10, Lys35, Thr39, Thr70, Glu97, His103 and Phe104, appear to occupy similar locations and are likely to play similar roles in protein/ligand interaction. However, the orientation and spatial arrangement of these residues are far from identical with those observed in RNase A. To further identify the speci®c binding sites, we have obtained 2D NOESY spectra of RC-RNase in the presence of excess inhibitors, 20 ,50 -CpG and 30 ,50 -dCpdG, in 2H2O. Table 4 summarizes the observed NOE cross-peaks between RC-RNase and the two inhibitors. Although the proton resonances of cytosine base appear to experience the largest chemical shift changes, the only observable NOE cross-peaks are between H10 and H8 protons of the guanosine and the side-chains of Val37 and Thr70 in the protein moiety. As shown in Figure 9, Val37 and Thr70 are near the B1 binding site. Thus, the B2 base binds to the B1 site, a con®guration expected for the retro-binding mode (Aguilar et al., 1989, 1991, 1992). Our observation provides the ®rst experimental evidence that such retro-binding mode does occur in solution conditions. It will be interesting to investigate whether such a binding mode is affected by the presence of a phosphate ion. It should also be cautioned that, due to the limited number of NOE cross-peaks between RC-RNase and the inhibitor are observable, this conclusion should be treated as qualitative and the exact binding con®guration needs to be determined more rigorously.

Materials and Methods Preparation of RC-RNase sample Extraction and puri®cation of RC-RNase from bullfrog ooctyes was accomplished as described (Liao et al., 1996;

240 Chen et al., 1996). Brie¯y, the oocytes were released from the ovaries (cut into approximately 4 cm3 pieces) by incubation with 0.15% (w/v) type II collagenase. The ribonuclease was puri®ed by precipitation of yolk granules, extraction of RC-RNase with 0.09 M NaCl, and selective removal of impurities by chromatography on phosphocellulose and carboxymethyl-cellulose columns. A yield of 150 mg of RC-RNase could be puri®ed from a mature female bullfrog (600 g in weight), which has 100 g of ovary tissue. The ribonuclease activity was assayed by the dinucleotide method (Liao, 1992). Samples for NMR experiments contained about 0.35 ml of 2 to 3 mM protein in 50 mM potassium phosphate buffer, pH 3.5 to 7.0. Buffer exchange and pH adjustments were achieved by repeated dilution with appropriate buffers and concentrating with Centricon ®lters. The pH of the ®nal ®ltrate was taken as the pH of the protein sample. Upon attaining the desired pH, 10% (v/v) of 2H2O was added. For monitoring the exchange rates of labile protons, the concentrated sample in H2O was lyophilized only once and re-dissolved in 2H2O (99.99% 2H) and NMR spectra were acquired immediately and thereafter at appropriate time intervals. For preparing the sample in 2H2O at pH* 7.56, the concentrated protein sample was repeatedly lylphilized and redissolved in 2H2O. pH values were measured with a JENCO microelectronic pH-vision model 6071 pH meter equipped with a 4 mm electrode. All reported pH values were direct readings from the pH meter without correction for isotope effect. pH* is used to indicate a direct pH meter reading of 2H2O solutions uncorrected for the deuterium isotopic effect using electrodes standardized in H2O buffers. Cytidylyl(20 ! 50 )guanosine (20 ,50 CpG) and 20 -deoxycytidylyl(30 ! 50 )-20 -deoxyguanosine (30 ,50 dCpdG) were purchased from Sigma (St. Louis, MO) and were used without further puri®cation. The inhibitors were added directly to the protein sample to an inhibitor to protein ratio of 2:1 or 4:1 at pH* 7.56 in 50 mM phosphate buffer. NMR spectroscopy All NMR spectra were recorded at 600.13 MHz on Bruker AVANCE-600 or AMX-600 spectrometers (Karlsruhe, Germany). For DQF-COSY spectra, 700 to 1024 t1 increments were collected, each with 16 to 32 scans of 2 k or 4 k complex data points (Rance et al., 1983). The TOCSY spectra were collected with 512 to 700 t1 increments with 16 transients of 2 k complex points (Braunschweiler & Ernst, 1983; Bothner-By et al., 1984; Bax & Davis, 1985). We used MLEV-17 spin-lock sequence of 18 and 55 ms in 2H2O media and 18, 32, 45 and 55 ms for protic media. For NOESY spectra, 512 to 700 2K complex FIDs increments, each with 32 scans averaging were collected (Kumar et al., 1980; Bodenhausen et al., 1984). Spectra of mixing times of 50, 100 and 200 ms were recorded in time-proportioned phase sensitive (TPPI) mode (Red®eld & Kunz, 1975; Marion & WuÈthrich, 1983). Water suppression was achieved by 1.4 seconds presaturation at the water frequency, or by the gradient method (Piotto et al., 1992). To help resolving spectral overlapping, data were collected at several temperatures; 298, 300, 305 and 320 k for the pH 3.5 samples and 305 k for the pH* 7.56 samples and all protein/inhibitor complexes. Data were transferred to SGI workstations (Silicon Graphics, CA, USA) for all processing and further analysis using Bruker UXNMR and AURELIA software packages (Karlsruhe, Germany). All datasets acquired were zero-®lled to

Solution Structure of a Cytotoxic Ribonuclease equal points in both dimensions prior to further processing. A 60 -shifted skewed sine bell window function was used in all NOESY and TOCSY spectra, and a 20 or 30 -shifted skewed sine bell function was used for all COSY spectra. DSS was used as chemical shift reference. Interproton distance restraints The distance restraints were primarily determined from a NOESY spectrum (mixing time 100 ms) of RC-RNase obtained at 310 k, pH 3.5. In the ®rst stage of calculation, the inter-proton distances were assigned 1.8 Ê , 1.8 to 3.5 A Ê , 1.8 to 4.5 A Ê or 1.8 to 5.5 A Ê , respectto 2.5 A ively, depending on whether the NOE intensities are strong, medium, weak or very weak. These distances were calibrated using a calibration curve based on the known distances of methylene protons and aromatic protons. In all, 150 structures generated at this ®rst run of simulated annealing were examined for serious violations and goodness of ®t for each proton pair with observable NOE cross-peaks. The distance restraints for those showing severe distance violations were either relaxed or removed from the data base. On the other hand, the distance restraints for those with good ®ts were narrowed This process was repeated for ten cycles. In the ®nal cycle of simulated annealing calculation, more Ê , 2.8 to 3.5 A Ê, restricted distance restraints of 2.2 to 2.8 A Ê and 3.5 to 5.5 A Ê are used for strong, med3.2 to 4.5 A ium, weak and very weak NOE cross-peaks, respectively (Metzlr et al., 1992; MuÈhlhahn et al., 1994). For severely overlapping resonances, NOESY spectra obtained at different temperature and pH conditions were used to assist the assignment of the intensity class. Ambiguous NOE cross-peaks with several possible assignments were ®rmly assigned based on structures generated at intermediate stages of re®nement using back-calculation. The process was repeated until no new ambiguity was identi®ed. New assignments were obtained based on this iterative process. Pseudo-atom restraints were used when the stereospeci®c protons were not assigned. Torsion angle restraints and stereospecific assignment The 3JNHa coupling constants were estimated from the residual intensity of the anti-phase cross-peak in DQFCOSY spectra (Rance et al., 1983) recorded in H2O. Torsion restraints of f of ÿ120(40) for 3JHNa greater than 8 Hz and ÿ60(30) for 3JHNa smaller than 6 Hz were used for structure calculation in the early stage. The stereospeci®c assignments were derived using the established methods (Wagner et al., 1987; GuÈntert et al., 1989; Cai et al., 1995). The 3Jab and 3Jab, coupling constants were estimated as either large or small based on: (i) the intensities of the cross-peaks observed in a DQF-COSY spectrum in 2H2O and a TOCSY spectrum recorded in short mixing time (18 ms); and (ii) the relative intensities of the intra-residue CaH-CbH and NH-CbH NOE crosspeaks. The stereospeci®c assignment of b-methylene also allowed us to assign the w1 torsion angle restraints to either 60(30) , 180(30) or ÿ60(30) . Fifteen prochiral assignments were made with certainty, and these assignments provide better distance restraints for over 80 NOE cross-peaks. The prochiral assignments were found to be in total agreement with the structures generated in the early stage of calculation. w1 restraints were used in later stage of calculation. Stereospeci®c assignments of the valine methyl resonances were obtained

241

Solution Structure of a Cytotoxic Ribonuclease from the 3Jab coupling constants and the NOE cross-peak intensities between the valine methyl protons and its NH protons (Zuiderweg et al., 1985). Hydrogen bond and disulfide restraints The amide proton exchange rates were identi®ed from residual amide proton signals observed in several TOCSY spectra recorded at 310 K at pH 3.5. The ®rst spectrum was recorded within ®ve hours after the lyophilized sample was redissolved in 2H2O. The last TOCSY spectrum, as shown in Figure 10, was obtained one week after dissolving the sample in 2H2O to further identify the very slowly exchanging NH protons. The amide proton exchange rates were categorized into three classes; fast, intermediate and slow exchange rates (Chen et al., 1996). Hydrogen bond formation or solvent exclusion from the amide protons were assumed to account for the slow and intermediate exchange amide protons. From sequential and inter-strand NOEs, most of these protons have been identi®ed to be associated with particular secondary structures (Chen et al., 1996). For better convergence, those slowly exchanging protons observed to be part of the b-sheet inter-strand NHi-NHj, NHiCaHj ‡ 1 and CaHi ÿ 1-CaHj ‡ 1 NOE network were included as spatial restraints in the latter stages of structure calculation. The O-N distance was set to be between Ê , and the distance between the NH proton 2.8 and 3.3 A and the carbonyl oxygen atoms was set to be between Ê . The disul®de bonds used in the structure 1.8 and 2.3 A

Figure 10. Fingerprint region of a TOCSY spectrum showing the very slowly exchanging amide protons. The spectrum was obtained at 310 K with a 2 mM protein sample that had been redissolved in 2H2O for a week after it was lyophilized from a H2O solution containing 50 mM phosphate, pH 3.5.

calculation are Cys19-Cys71, Cys34-Cys81, Cys52-Cys96 and Cys93-Cys110. The disul®de bond restraints were Ê for the distance between the set as follows: 1.96 to 2.07 A Ê for the distance between two sulfur atoms; 2.9 to 3.37 A the sulfur atom of one cysteine residue and the b carbon Ê for atom of the other cysteine residue; and 3.89 to 3.98 A the distance between the two b carbon atoms.

Tertiary structure calculations All structure and dynamical simulated annealing calculations (Nilges et al., 1988) were carried out with the program X-PLOR 3.1 (BruÈnger, 1992) on a SGI Indigo II workstation. The total target function Ftot contains the following terms: Ftot ˆ Fbond ‡ Fangle ‡ Fimproper ‡ Frepel ‡ FNOE ‡ Ftor where Fbond, Fangle, Fimproper and Ftor are the target functions for maintaining correct bond lengths, angles, planes and chirality, respectively. The force constants of the energy terms for bonds, angles, and impropers were set to the uniform value of 800 kcal molÿ1 during the simulated annealing stage. The non-bonded interactions, Frepel, are represented by a simple van der Waals term with variable force constant, krepel (Nilges et al., 1988). The initial value of krepel is very low (0.002 kcal molÿ1 Ê ÿ4), and is multiplied by a factor of 1.4 prior to each A Ê ÿ4. The NOE cycle to its ®nal value of 1.0 kcal molÿ1 A distance constraints, FNOE, are represented by a softsquare potential with a variable force constant kNOE (Clore et al., 1986). The value of kNOE was doubled at the beginning of each cycle, increasing from its initial value Ê ÿ2 to its ®nal values of 100 kcal molÿ1 of 10 kcal molÿ1 A Ê ÿ1. The force constant for the square well torsion potenA tial term, Ftor, was set to either 80 or 60 kcal molÿ1 degÿ2 depending on the accuracy of the measured coupling constants. Neither electrostatic nor hydrogen bond potential was used during structure calculation. Initially, an extended structure with the correct amino acid sequence was used as the input for a protocol to generate 50 starting structures by randomizing the f and c angles of all residues in the template sequence. These structures were subjected to 15 ps of molecular dynamics treatment at 1000 K prior to the introduction of an S-S bond at the energy minimization stage following the ®rst annealing cycle. A homology structure was built by aligning the sequence of RC-RNase, onconase and RNase A, and deciding the structure of a particular segment of RC-RNase. The structure of a particular segment of RCRNase is assigned the corresponding structure of either onconase or RNase A, if the sequence of that segment is very similar to either onconase or RNase A. Otherwise, the structure of RC-RNase was assigned the average structure of onconase and RNase A. The structure of RCRNase built in this way was further energy minimized to obtain the ®nal homology structure. The 50 randomly generated structures, together with this homology structure, were used as the starting structures for subsequent rounds of simulated annealing calculations. Ten cycles of simulated annealing calculations were carried out, with distance restraints narrowed and poorly ®tted NOE cross-peaks removed from the restraint list, as described in the previous section. At the end of each stage, several structures were examined to evaluate NOE violations, hydrogen bond formation and stereospeci®c assignments. The re®ne routine was used for ®nal structure optimization.

242 Structure analysis The structures were analyzed and examined with X-PLOR (BruÈnger, 1992), PROCHECK-NMR (Laskowski et al., 1993, 1996), RIBBONS (Carson, 1987, 1991), GRASP (Nicholls et al., 1991) and InsightII (Molecular Simulation Inc.) programs. Coordinates for onconase (Mosimann et al., 1994; 1ONC) and bovine pancratic RNase A (Santoro et al., 1993; 2AAS) were taken directly from the Brookhaven Protein Data Bank. For calculating the surface charge, we employ the default partial charge table (full.crg) supplied with the GRASP program.

Acknowledgements We thank Dr Yao-Di Liao for technical assistance and helpful suggestions. This work was supported by the National Science Council of the Republic of China (NSC86-2314-B-001-031) and the Structural Biology Program of the Academia Sinica, Taiwan, the Republic of China.

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Edited by P. E. Wright (Received 4 May 1998; received in revised form 13 July 1998; accepted 13 July 1998)