Promotion of Purine Nucleotide Binding to Thymidylate Synthase By

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3493-3497, April 1995 Biochemistry

Promotion of purine nucleotide binding to thymidylate synthase by a potent folate analogue inhibitor, 1843U89 (CD spectra/stacking of folate analogue with nucleotide/helix shift at active site/conformation change/x-ray crystallography) ANDRZEJ WEICHSEL*, WILLIAM R. MONTFORT*, JAROSLAW CIESLAt, AND FRANK MALEYtt *Department of Biochemistry, University of Arizona, Tucson, AZ 85721; and tWadsworth Center, New York State Department of Health and Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, NY 12201-0509 Communicated by Gertrude B. Elion, Burroughs Wellcome, Research Triangle Park NC, January 5, 1995 (received for review November 7, 1994)

ABSTRACT A folate analogue, 1843U89 (U89), with potential as a chemotherapeutic agent due to its potent and specific inhibition of thymidylate synthase (TS; EC 2.1.1.45), greatly enhances not only the binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and dUMP to Escherichia coli TS but also that of dGMP, GMP, dIMP, and IMP. Guanine nucleotide binding was first detected by CD analysis, which revealed a unique spectrum for the TS-dGMP-U89 ternary complex. The quantitative binding of dGMP relative to GMP, FdUMP, and dUMP was determined in the presence and absence of U89 by ultrafiltration analysis, which revealed that 1843U89 although the binding ofGMP and dGMP could not be detected in the absence of U89 both were bound in its presence. The Kd for dGMP was about the same as that for dUMP and FdUMP, with binding of the latter two nucleotides being increased by two orders of magnitude by U89. An explanation for the binding of dGMP was provided by x-ray diffraction studies that revealed an extensive stacking interaction between the guanine of dGMP and the benzoquinazoline ring of U89 and hydrogen bonds similar to those involved in dUMP binding. In addition, binding energy was provided through a water molecule that formed hydrogen bonds to both N7 ofdGMP and the 5,1O-CH2H4PteGlu hydroxyl of Tyr-94. Accommodation of the larger dGMP molecule was accomplished through a distortion of the active site and a shift of the deoxyribose moiety to a new position. FIG. 1. Structural comparison of U89 and 5,10-CH2H4PteGlu. These rearrangements also enabled the binding of GMP to in the nanomolar range (8) but was too toxic to be used as a occur by creating a pocket for the ribose 2' hydroxyl group, chemotherapeutic agent (9). overcoming the normal TS discrimination against nucleotides More recently, a benzoquinazoline derivative of folate, containing the 2' hydroxyl. 1843U89 (U89), which inhibited human TS in the subnano- molar range, was developed at Burroughs Wellcome (10, 11) The only clinically useful antitumor agent to date that exerts based on its similarity in structure to CH2H4PteGlu and PDDF its effect by inhibiting thymidylate synthase (TS; EC 2.1.1.45) (Fig. 1). Earlier studies (12) showed that TS formed charac- is 5-fluorouracil (1, 2). This is a result of fluorouracil's metabolic teristic CD patterns with dUMP or FdUMP and various folate conversion to 5-fluoro-2'-deoxyuridine 5'-monophosphate derivatives. In the course of examining U89's interaction with (FdUMP) (3) with the latter acting as an inhibitory transition TS by CD, we found that not only did FdUMP and dUMP yield state analogue when bound to TS in the presence of this characteristic ellipticity patterns with this folate analogue but enzyme's second substrate 5,10-methylenetetrahydrofolate unexpectedly so did GMP, dGMP, and dIMP. In this report, we (CH2H4PteGlu) (4, 5). Due to the lack of specificity of this present evidence from crystallographic studies that explains inhibition against normal and tumor tissue TS, the resulting how U89 promotes the formation of ternary complexes with severe toxicity associated with the use of fluorouracil limits its TS and selective purine nucleotides.§ usefulness. Attention has therefore been focused on developing folate MATERIALS AND METHODS analogues that might be more selective and as a consequence Enzyme Preparation. ThyA-TS was amplified using a high- more effective. Evidence in support of this possibility was expression system that yielded 40-50% of the cellular protein suggested from the finding that folylpolyglutamates under certain conditions could inhibit T4 with selectively phage-TS Abbreviations: FdUMP, 5-fluoro-2'-deoxyuridine 5'-monophosphate; minimal effects on Escherichia coli TS (6). The corresponding U89, 1843U89; TS, thymidylate synthase; H2PteGlu, dihydrofolate; methotrexate polyglutamates were also effective TS inhibitors H4PteGlu, tetrahydrofolate; CH2H4PteGlu, 5,10-methylenetetrahy- (7). However, the first really potent inhibitory folate analogue, drofolate; PDDF, lo-propargyl 5,8-dideazafolate. CB3717 (10-propargyl 5,8-dideazafolate; PDDF), inhibits TS 4To whom reprint requests should be addressed at: Wadsworth Center, New York State Department of Health, Empire State Plaza, P.O. Box 509, Albany, NY 12201. The publication costs of this article were defrayed in part by page charge §The atomic coordinates have been deposited in the Protein Data payment. This article must therefore be hereby marked "advertisement" in Bank, Chemistry Department, Brookhaven National Laboratory, accordance with 18 U.S.C. §1734 solely to indicate this fact. Upton, NY 11973 (reference 1TLC). 3493 Downloaded by guest on September 25, 2021 3494 Biochemistry: Weichsel et aL Proc. Natl. Acad Sci. USA 92 (1995) of E. coli as TS (F.M. and L. Changchien, unpublished data). other folate derivatives. However, what was completely unex- The enzyme was purified to homogeneity on DE-52 basically pected was the formation of a CD pattern with dGMP (Fig. 2) as described, but with omission of the affinity column step (13). since there have been no data to suggest that purine nucle- CD Analyses. CD spectra were taken at room temperature otides could bind to or inhibit TS. The specific nature of this with a Jasco (Easton, MD) J-720 spectropolarimeter equipped reaction was revealed by the fact that neither dAMP nor with software to suppress noise and to develop difference deoxyguanosine 3'-monophosphate yielded a CD spectrum spectra. Circular 1-cm path-length cells were used that held 3 with U89 and TS. Not only did dGMP provide a spectrum with ml of solution. The concentration of components in this U89 and TS but so also did GMP, IMP, and dIMP (data not solution was thyA-TS, 2.2 AtM; FdUMP or dGMP, 18.3 ,tM; shown) with all differing somewhat at the low end of the UV U89, 6.1 ,uM; potassium phosphate (pH 7.5), 25 mM; MgCl2, spectrum from that obtained with FdUMP (Fig. 2). The extent 5 mM; and 2-mercaptoethanol, 20 mM. The spectra presented to which U89 promoted the binding of each nucleotide, are difference spectra obtained by subtracting the spectra from particularly GMP and dGMP, was measured using an ultra- the above solutions minus U89 from those containing this filtration assay (23) due to the greater sensitivity and precision compound. U89 was generously provided by Robert Ferone (Burroughs Wellcome). of this method relative to CD titration studies. X-Ray Crystallography Studies. Hexagonal crystals of the Nature of the Binding Affected by U89. It is apparent from ternary complex, TS-dGMP-U89, were grown in the dark using the results presented in Table 1 that U89 profoundly affects the the hanging drop method. A solution consisting of TS (22 binding of dUMP and FdUMP, which in both cases was mg/ml) 5 mM dGMP, 0.5 mM U89, 20 mM potassium increased by at least two orders of magnitude. Even more phosphate (pH 8.0), and 5 mM dithiothreitol was mixed 1:1 significant was the dramatic increase in the binding of dGMP with the precipitant solution [56% saturated (NH4)2SO4, 20 promoted by U89 since dGMP binding could not be detected mM potassium phosphate (pH 8.0), and 5 mM dithiothreitol] in its absence. The fact that GMP elicited a CD pattern is and equilibrated at room temperature against the precipitant consistent with the improvement in its binding, which was still solution. The resulting crystals belonged to space group P63 about 30-fold less than that of dGMP. The enhancement of and were isomorphous with those of the TS-dUMP-PDDF binding of nucleotides by folates and folylpolyglutamates and ternary complex (14) and TS-dUMP-U89. A single crystal vice versa is in agreement with earlier binding studies (24, 25). (0.34 x 0.22 x 0.38 mm) was used for data collection em- Of interest is the fact that only about one binding site per dimer ploying an Enraf Nonius diffractometer equipped with a FAST of E. coli TS was found for dUMP in the presence or absence detector (fast-scanning-area-sensitive television diffracto- of U89 while the binding of FdUMP was reduced from 2 to 1 meter) attached to a Cu anode x-ray source. Data reduction mol/mol of dimer by U89. The estimated number of binding was performed with software packages MADNES (15) and sites for dGMP and GMP was also about one per dimer. These PROCOR (16). Reflections were scaled with FBSCALE (17) and results are similar to those obtained for dUMP with U89 and structure factors and electron density maps were calculated human TS (26). Assuming that the human, L. casei, and E. coli using CC4 (18). The structure of TS-dUMP-U89 (A.W., un- TSs have similar binding characteristics, it appears that the published data) was used as a starting model for refinement binding of dUMP to the second site in the dimer is much with GPRLSA (19) and local rebuilding using FRODO (20) modified weaker than the first, resulting in an appearance of asymmetry to run on an Evans and Sutherland ESV workstation kindly in binding. Similar results have been obtained for specific provided by Florante Quiocho (Department of Biochemistry, folates and their analogues (24-27). Unlike dUMP, FdUMP Baylor College of Medicine, Houston). Figs. 3-5 were pro- binding while asymmetric (24) clearly binds to both sites, but duced with the program MOLSCRIPT (21). in the presence of specific folates its' binding to both sites is Ultrafiltration Studies. A procedure similar to that of Ormo greatly enhanced (24, 26, 28). Similarly, folate analogues such and Sjoberg (22) was used to measure Kd and binding site as D1694GIu4 and PDDFGlu4 bind to both active site regions, number for the various nucleotides employed. The resulting while CH2H4PteGlu4 and U89 prefer one of the two sites, data were analyzed by the method of Scott (23). The buffer particularly when dUMP is present (26). In those cases where employed for these studies contained 50 mM Tris HCI (pH binding to only one site is apparent, this may be a consequence 7.1), 6.7 mM dithiothreitol, and 10% ethylene glycol. The con- of the second site being subject to ligand-induced negative centrations of nucleotides and enzyme used were 0.5-40 ,uM cooperativity (26), as a consequence of the conformational [8-3H]GMP (75-550 dpm/pmol) and 1.3 ,uM TS; 0.04-1.7 changes associated with ,uM [8-3H]dGMP (235 dpm/pmol) and 0.48 ,uM TS; 0.2-1.3 ligand binding to TS (14). ,uM [6-3H]dUMP (270-4200 dpm/pmol) and 0.05-11 ,uM TS; 0.02-5 ,uM [6-3H]FdUMP (970-28,100 dpm/pmol) and 0.2 107r ,uM TS. When U89 was present its concentration was varied from 6 to 10 ,uM. The solutions were incubated at room temperature for 20 min and then centrifuged in UFC3TTK poly- sulfone filters from Millipore for 45 sec at 5000 x g with an Eppendorf centrifuge. The labeled nucleotides were pur- chased from Moravek Biochemicals (La Brea, CA). RESULTS CD Analysis of Ternary Complexes. Previous studies (12) suggested that dUMP and FdUMP evoked ellipticity patterns with Lactobacillus casei TS in the presence of tetrahydrofolate (H4PteGlu), dihydrofolate (H2PteGlu), and CH2H4PteGlu that were characteristic of the folate employed. The difference in the ellipticity patterns appears to result from the manner in 1 which dUMP or FdUMP stacks with the folate analogue at the active site of TS (14). In the course of similar studies with U89, Wavelength, nm it was evident that FdUMP and dUMP yielded strikingly FIG. 2. Comparison of CD patterns generated by FdUMP and different ellipticity patterns with U89 than those provided by dGMP in the presence of U89 and TS. See text for details. Downloaded by guest on September 25, 2021 Biochemistry: Weichsel et al Proc. Natl. Acad Sci USA 92 (1995) 3495 Table 1. Effect of U89 on nucleotide binding to E. coli TS U89. The positions of ligands and amino acids lining the active U89 sites did not differ appreciably between the two monomers. This apparent disparity with the results for the binding studies Absence Presence presented above was likely due to the difference in ligand Ligand Kd, ,uM n* Kd, ,uM n* concentrations used in the two sets of experiments, since the crystallizations were conducted in the presence of saturating dUMP 3.2 ± 0.80 1.2 ± 0.45 0.040 ± 0.002 1.2 ± 0.06 ligand concentrations while the solution studies were carried FdUMP 9.2 ± ± ± ± 0.92 2.0 0.32 0.013 0.002 0.8 0.15 out under limited ligand concentrations. E. coli and L. casei TS dGMP ND - 0.076 ± 0.02 1.0 ± 0.06 have now been crystallized in six different crystal forms and in GMP ND 2.7 ± 0.26 1.1 ± 0.20 the presence of a wide variety of ligands and in all cases have ND, not detectable; see text for experimental details. been found to contain ligands in both active sites (refs. 14, 29, *Number of binding sites per mol of TS; mean value ± SD from two 32, and 33; A.W. and W.R.M., unpublished data). to four experiments. The position of dGMP in the TS active site was quite similar X-Ray Diffraction Studies. To understand how TS binds to that of dUMP in the TS-dUMP-U89 and TS-dUMP-PDDF dGMP in the presence of U89, the crystal structure of the structures but required local shifts in the active site region to ternary complex was determined. Ligand positions were achieve this position (Fig. 3). Accommodation of the larger clearly interpretable in the initial electron density maps as dGMP molecule was accomplished through a 0.4-A shift of the J helix (residues away from the a were differences from the dUMP-containing model structure. 173-193) active site, 0.8-A Conventional model building and crystallographic refinement shift of the deoxyribose moiety in the direction opposite to that of the J helix, and a shift of the Gln-19-Thr-24 loop of about were sufficient to produce the model described below, which 0.5 A outward from the active site. Arg-21, which is part of this was determined to a nominal resolution of2.1 A, with all amino loop and bound to the dGMP phosphate, required an altered acids and ligands clearly defined in the final electron density conformation to maintain contact with the nucleotide in this map. Table 2 lists data relevant to the structure determination. new position. Likewise, Tyr-209 required a 5° rotation to The overall topology of the structure was typical of TS remain hydrogen bound to the ribose 3' hydroxyl. ternary complex structures (14, 29-33). The protein displayed U89 in the TS-dUMP-U89 ternary complex contacted res- an extensive conformational change as compared with the idues on both sides of the active site. To maintain these unliganded structure, similar to that previously seen for the contacts in the dGMP-containing complex required movement complex with the smaller ligands dUMP and PDDF (14). of both U89 and residues contacting U89. U89 remained in While the overall conformational change remained the same, contact with the J helix by translating -0.4 A in the same considerable local changes in the active site were required to direction as the helix. On the other side of the active site a accommodate the binding of the larger U89 ligand. These similar shift took place in the 11-amino acid segment His-73- shifts included displacement of the Leu-72-Pro-92 peptide Trp-83, which allowed Ile-79, Trp-80, and Trp-83 to remain in fragment, a shift of the Cys-50-Ile-55 loop, a shift of the contact with the ligand. The only other difference between the C-terminal fragment outward from the active site, and a dUMP- and dGMP-containing structures was a slight change difference in the conformation of Phe-176. These local in the position of Arg-49-Leu-52, which was likely due to changes were not transferred to the rest of the protein mole- 2-mercaptoethanol oxidation of Cys-50 in the dUMP-containing cule and will be discussed with the TS-dUMP-U89 structure in structure. This difference did not appear to alter the position of a separate paper (A.W. and W.R.M., unpublished data). The U89. The His-51 and Arg-53 side chains also differed between the residue numbers used in this paper are for E. coli TS. When converting to L. casei TS the numbering is as follows: I + 2 for I 5 87, I + 52 forI > 87. The asymmetric (unique) portion of the crystals contained the full TS dimer, which allowed for an independent evaluation of each monomer. dGMP and U89 were found to be well N177 ordered in both active sites except for the glutamate portion of Table 2. Crystallographic data for TS-dGMP-U89 Space group P63 Unit cell, A a = b = 127.14(2), c = 67.86(1) a = a = 90', y = 120° Resolution, A 15-2.1 Unique/total reflections 34,323/120,983 Completeness, % 94 Rsym* 0.086 Rcrystt 0.18 rms deviation bonds, A 0.018 rms angles, deg 2.0 No. of included water molecules 169 Average temperature factors for subunits I and II, A2 Monomer I/II 22/24 dGMP I/II 11/16 U89 I/II 30/37

- FIG. 3. Overlap of dUMP and dGMP within the active site of TS, *RsymRym=hkl= , Yi I'avg 1 I/ hkl7 IavgI I. when present as their respective ternary complexes with U89. This tRcryst = -I Fobs - Fcalc 1/E Fobs I. figure was produced with the program MOLSCRIPT (21) as were Figs. 4 hkl hkl and 5. Downloaded by guest on September 25, 2021 3496 Biochemistry: Weichsel et at Proc. Natl. Acad Sci. USA 92 (1995) two monomers in the dGMP-containing structure, again without effect on ligand position. The active site residues involved in the binding of dUMP (Fig. 4A) are similar to those involved in the binding of dGMP (Fig. 4B). Hydrogen bonds to the dGMP deoxyribose and phosphate groups are unchanged. As can be seen in Fig. 3, the hydrogen bonding surface presented by guanine was very similar to that of uracil, and hydrogen bonds to the pyrimidine portion of dUMP (Asn-177 and Asp-169) form similar hydrogen bonds with the purine portion of dGMP (Fig. 4B). The 2-amino group of guanine was not involved in hydrogen bonding but was in van der Waals contact with Gly-173. A new hydrogen bond not present in the dUMP structure was mediated by a water molecule between N7 of dGMP and the Tyr-94 hydroxyl. A water molecule was also bound to Tyr-94 in the dUMP-containing structure but was not hydrogen bound to the nucleotide. In contrast, a water-mediated hydrogen bond between 04 of dUMP and His-147 is lost in the TS-dGMP-U89 structure. A striking feature of the ternary complex was the extensive stacking between dGMP and U89. The purine ring of dGMP FIG. 5. Stacking interaction of dGMP (open bonds) and U89 (filled is located parallel to the benzoquinazoline ring of U89 at a bonds) in the TS-dGMP-U89 structure. Nitrogen atoms, large open distance of 3.5 A (Fig. 5) and favorable van der Waals contacts spheres; carbon atoms, small open spheres; oxygen, shaded spheres. were gained with N7, C8, and N9 of dGMP as compared with dUMP, which does not contain these atoms. The unique unpublished data). The fact that U89 yielded a characteristic stacking of the purine and pyrimidine rings with the ring CD pattern with FdUMP and TS was not unexpected, but to structures of the folates and their analogues is undoubtedly yield spectra with dGMP (Fig. 2), GMP, and IMP (not responsible for their unique CD spectra. presented) was indeed a surprise since there has been no evidence to suggest that purine nucleotides bind to TS in the presence or absence of folates. The specificity of this reaction DISCUSSION is revealed by the fact that deoxyguanosine 3'-monophosphate The primary function of TS in the cell is to convert dUMP and dAMP did not yield spectra, nor did dCMP and UMP. to dTMP so that the latter essential nucleotide can participate Preliminary studies indicate that dGMP can also interact or in the synthesis of DNA. The mechanism of dTMP synthesis stack with PDDF and D1694 to yield their characteristic is particularly intriguing since the second substrate, spectra but the signal appears to be weaker than with U89. CH2H4PteGlu, provides both the methylene group and a Those forces that enable the guanine nucleotides to bind are hydride ion from the C6 position of the pteridine ring (34) in clearly revealed by examining the crystal structure of the one of the more unique reactions in nature. To effect this TS-dGMP-U89 ternary complex. The binding of dGMP is reaction, the substrates on binding to TS introduce a marked similar to that of dUMP (Fig. 3), and similar conformations were change in the protein (14, 30), which brings the substrates in induced in TS on forming both ternary complexes. The purine proper alignment with groups on the protein so as to enable of dGMP forms hydrogen bonds to Asn-177 and Asp-169 that catalysis to occur. Substrate analogues such as FdUMP and are nearly identical to those formed by dUMP (Fig. 4), and PDDF, when added to TS, effect a similar structural alteration, binding of ribose and phosphate involves the same amino acids as does PDDF alone (31). While such conformational transi- in both cases. However, a water-mediated hydrogen bond not tions are clearly seen in x-ray diffraction studies, they were observed in TS-dUMP-U89 was formed between N7 of dGMP suspected much earlier in CD analyses that revealed that folate and the hydroxyl of Tyr-94, whereas a water-mediated hydro- derivatives yielded characteristic ellipticity patterns in the gen bond between dUMP and His-147 was lost. Accommoda- presence of FdUMP and L. casei TS (12). More recently, these tion of the larger purine ring in the dGMP-containing struc- studies were expanded with folate-based inhibitors of TS, ture was accomplished through several shifts in the active site including PDDF, U89, and other analogues, each of which of amino acids in contact with the nucleotide. These included provided a unique difference spectrum when complexed with a 0.4-A shift of the J helix, a 0.8-A shift of the dGMP dUMP (or FdUMP) and thyA-TS (F.M. and L. Changchien, deoxyribose, a 5° rotation in the Tyr-209 side chain, and a shift

A N1B< Y94 B :N H147 FIG. 4. Hydrogen bond and in- x,~ *¶?0w Ateratomic distances between nucle- 'W@ ' Ayg4 otide (filled bonds) and specific Y94 .t avamino acids (open bonds) in the ternary complexes of TS-dUMP-U89 (A) and TS-dGMP-U89 (B). Oxy- gens (including those belonging to solvent), filled circles; carbons, small open circles; nitrogens, large open circles. Hydrogen bonds, long- dashed lines; van der Waals contacts between Gly-173 and dGMP, short- dashed lines. The corresponding distances between Gly-173 and dUMP are presented for comparison. All distances are in angstroms. Downloaded by guest on September 25, 2021 Biochemistry: Weichsel et at Proc. Natl. Acad. Sci. USA 92 (1995) 3497 of about 0.5 A of the loop containing Arg-21. These shifts 4. Langenbach, R. J., Danenberg, P. V. & Heidelberg, C. (1972) served to open a gap near the 2' position of the dGMP ribose. Biochem. Biophys. Res. Commun. 48, 1565-1571. The binding of GMP was unexpected but can be explained 5. Santi, D. V. & McHenry, C. S. (1972) Proc. Natl. Acad. Sci. USA 69, 1855-1857. based on comparison of the dUMP- and dGMP-containing 6. Maley, G. F., Maley, F. & Baugh, C. M. (1979)J. Biol. Chem. 254, structures. In the dUMP-containing ternary complex the ribose 7485-7487. 2' carbon is in van der Waals contact with residues 167-169, 7. Kisliuk, R. L., Gaumont, Y., Baugh, C. M., Galivan, J. H., Maley, which form a small pocket in the active site. These contacts G. F. & Maley, F. (1979) in Developments in Biochemistry, eds. produce discrimination between dUMP and UMP as potential Kisliuk, R. L. & Brown, G. M. (Elsevier/North Holland, Am- substrates for the enzyme (14). The gap formed near the 2' sterdam), Vol. 4, pp. 431-435. position in the dGMP-containing structure provides space for 8. Jones, T. R., Calvert, A. H., Jackman, A. L., Brown, S. J., Jones, the 2' hydroxyl of GMP. When GMP was modeled into the M. & Harrap, K. R. (1981) Eur. J. Cancer 17, 11-19. 9. Alison, D. L., Newell, D. R., Sessa, C., Harland, S. J., Hart, dGMP-containing structure, the hydroxyl filled this gap but L. I. I., Harrap, K. R. & Calvert, A. H. (1985) Cancer Chemother. retained a slight steric conflict with the main-chain nitrogens Pharmacol. 14, 265-271. of Cys-168 (2.9 A) and Asp-169 (2.8 A). These remaining 10. Pendergast, W., Dickerson, S., Dev, I., Ferone, R., Duch, D. & contacts can probably be relieved through additional shifts in Smith, G. (1992) Proc. Am. Assoc. Cancer Res. 33, 47 (abstr.). either ligand or protein and are probably responsible for the 11. Duch, D. S., Banks, S., Dev, I. K., Dickerson, S. H., Ferone, R., decreased binding of GMP relative to dGMP. Thus it appears Heath, L. S., Humphreys, J., Knick, V., Pendergast, W., Singer, that the shift in the active site to accommodate the larger S., Smith, G. K., Waters, K. & Wilson, H. R. (1993) Cancer Res. 53, 810-818. purine ring of dGMP partially removes the steric restrictions 12. Galivan, J. H., Maley, G. F. & Maley, F. (1975) Biochemistry 14, on ribose binding. 3338-3344. The question of whether one or two binding sites are 13. Maley, G. F. & Maley, F. (1988) J. Biol. Chem. 263, 7620-7627. involved in substrate or analogue binding is still a rather 14. Montfort, W. R., Perry, K. M., Fauman, E. B., Finer-Moore, contentious one, as the values obtained appear related to a J. S., Maley, G. F., Maley, F. & Stroud, R. M. (1990) Biochemistry number of factors including buffer and folate analogue used as 29, 6964-6967. well as concentration of the nucleotide being studied. Most of 15. Messerschmidt, A. & Pflugrath, J. W. (1987) J. Appl. Crystallogr. the 20, 306-315. binding techniques employed indicate that dUMP binds 16. Kabsch, W. (1988) J. Appl. Crystallogr. 21, 916-934. primarily to one of the two TS substrate sites (24-26, 35, 36), 17. Weissman, L. (1982) in Computational Crystallography, ed. Sayre, with the second one being extremely weak even in the presence D. (Clarendon, Oxford), pp. 56-63. of folylpolyglutamates (26, 27). The results in Table 1 for dUMP 18. CCP4 (1979) SERC Collaborative Computing No. 4: Suite of binding are consistent with these findings even when U89 is Programs for Protein Crystallography (Daresbury Laboratory, added. The binding of FdUMP clearly reveals that both active Warrington, U. K.). sites contribute to its binding but in an asymmetric manner- 19. Furey, W., Wang, B. C. & Sax, M. (1982) J. Appl. Crystallogr. 15, that is, binding to one site is preferred over the other (24, 37, 160-166. 20. Jones, A. (1978) J. Appl. Crystallogr. 11, 614-617. 38). However, in the presence of U89 the binding is reduced 21. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950. to a single site that is comparable to that obtained with dUMP, 22. Ormo, M. & Sjoberg, B.-M. (1990)Anal. Biochem. 189, 138-141. dGMP, and GMP. Whether, as suggested by Dev et al (26), 23. Scott, R. L. (1956) Rec. Trav. Chim. Pays-Bas 75, 787-789. ligand-induced negative cooperativity plays a significant role 24. Galivan, J. H., Maley, G. F. & Maley, F. (1976) Biochemistry 15, in this process remains to be determined. The binding studies 356-362. contrast with those obtained on examining liganded TS crys- 25. Galivan, J. H., Maley, F. & Baugh, C. M. (1976) Biochem. tals by x-ray crystallography, where saturating ligand concen- Biophys. Res. Commun. 71, 527-534. trations are used and both active sites are filled. 26. Dev, I. K., Dallas, W. S., Ferone, R., Hanlon, M., McKee, D. D. & Yates, B. B. (1994) J. Biol. Chem. 269, 1873-1882. The significance of the toxicity of U89 to TS in vivo and its 27. Lockshin, A. & Danenberg, D. V. (1979) J. Biol. Chem. 254, promotion of guanine nucleotide binding remains to be de- 12285-12288. termined. However, since nucleotides enhance the binding of 28. Galivan, J., Maley, F. & Baugh, C. M. (1977) Arch. Biochem. folate and vice versa, it would be of interest to determine Biophys. 184, 346-354. whether guanine or other nucleotides in situ potentiate the 29. Matthews, D. A., Villafranca, J. E., Janson, C. A., Shith, W. W., activity of U89 and, if so, whether these nucleotides can be Welsh, K. & Freer, S. (1990) J. Mol. Biol. 214, 937-948. detected in association with TS in U89-treated cells. 30. Matthews, D. A., Appelt, K., Oatley, S. J. & Xuong, N. H. (1990) J. Mol. Biol. 214, 923-936. 31. Kamb, A., Finer-Moore, J. S. & Stroud, R. M. (1992) Biochem- We express our appreciation to Judith Valentino for her help in istry 31, 12876-12884. preparing this manuscript. We gratefully acknowledge the use of the 32. Fauman, E. B., Rutenber, E. E., Maley, G. F., Maley, F. & Wadsworth Center's Biochemistry Core Facility, which provided the Stroud, R. M. (1994) Biochemistry 33, 1502-1511. Jasco J-720 spectropolarimeter for the CD studies described in this 33. Finer-Moore, J., Fauman, E. 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