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Crystal Structures of Human Bifunctional Enzyme Aminoimidazole-4-carboxamide Ribonucleotide Transformylase/IMP Cyclohydrolase in Complex with Potent Sulfonyl-containing Antifolates

2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês

10.1074/jbc.m313691200

ISSN

1083-351X

Autores

Cheom‐Gil Cheong, Dennis W. Wolan, S.E. Greasley, P. Horton, G. Peter Beardsley, Ian A. Wilson,

Tópico(s)

Protein Structure and Dynamics

Resumo

Aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (ATIC) is a bifunctional enzyme with folate-dependent AICAR transformylase and IMP cyclohydrolase activities that catalyzes the last two steps of purine biosynthesis. The AICAR transformylase inhibitors BW1540 and BW2315 are sulfamido-bridged 5,8-dideazafolate analogs with remarkably potent Ki values of 8 and 6 nm, respectively, compared with most other antifolates. Crystal structures of ATIC at 2.55 and 2.60 Å with each inhibitor, in the presence of substrate AICAR, revealed that the sulfonyl groups dominate inhibitor binding and orientation through interaction with the proposed oxyanion hole. These agents then appear to mimic the anionic transition state and now implicate Asn431′ in the reaction mechanism along with previously identified key catalytic residues Lys266 and His267. Potent and selective inhibition of the AICAR transformylase active site, compared with other folate-dependent enzymes, should therefore be pursued by further design of sulfonyl-containing antifolates. Aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (ATIC) is a bifunctional enzyme with folate-dependent AICAR transformylase and IMP cyclohydrolase activities that catalyzes the last two steps of purine biosynthesis. The AICAR transformylase inhibitors BW1540 and BW2315 are sulfamido-bridged 5,8-dideazafolate analogs with remarkably potent Ki values of 8 and 6 nm, respectively, compared with most other antifolates. Crystal structures of ATIC at 2.55 and 2.60 Å with each inhibitor, in the presence of substrate AICAR, revealed that the sulfonyl groups dominate inhibitor binding and orientation through interaction with the proposed oxyanion hole. These agents then appear to mimic the anionic transition state and now implicate Asn431′ in the reaction mechanism along with previously identified key catalytic residues Lys266 and His267. Potent and selective inhibition of the AICAR transformylase active site, compared with other folate-dependent enzymes, should therefore be pursued by further design of sulfonyl-containing antifolates. Cancer is responsible for 25% of all deaths within the United States, making it one of the leading causes of mortality second only to heart disease (1American Cancer Society Cancer Facts and Figures for 2002. Publisher, National Media Office, New York2002Google Scholar). As multiple metabolic pathways are implicated in disease initiation and progression, substantial efforts have been focused on inhibition of pathways that would either limit the spread of or completely eradicate tumors. In particular two enzymes, glycinamide ribonucleotide (GAR) 1The abbreviations used are: GAR, glycinamide ribonucleotide; Tfase, transformylase; AICAR, aminoimidazole-4-carboxamide ribonucleotide; ATIC, AICAR transformylase/IMP cyclohydrolase; 10-f-THF, N10-formyl-tetrahydrofolate; MSA, multisubstrate adduct inhibitor; BW2315, BW2315U89UC; BW1540, BW1540U88UD; XMP, xanthosine 5′-monophosphate; β-DADF, 2-[4-((2-amino-4-oxo-3,4-dihydropyrid-o[3,2-d]pyrimidin-6-ylmethyl)-{3-[5-carbamoyl-3-(3,4-dihydroxy-5-phosphonooxymethyltetrahydrofuran-2-yl) 3H-imidazol-4-yl]-acryloyl}-amino)benzoylamino]pentanedioic acid. transformylase (Tfase) and aminoimidazole-4-carboxamide ribonucleotide (AICAR) Tfase/inosine monophosphate (IMP) cyclohydrolase (ATIC), in the de novo purine biosynthesis pathway have been prime targets for chemotherapeutic development for two main reasons: 1) rapidly dividing tumors rely on purine de novo synthesis for production of adenine and guanine, whereas normal cells prefer the salvage pathway (2Jackson R.C. Harkrader R.J. Tattersall M.H.N. Fox R.M. Nucleosides and Cancer Treatment. Academic Press, Sydney1981: 18-31Google Scholar), and 2) both proteins are folate-dependent enzymes that can utilize the same cellular folate transport systems that facilitate entry of natural folates into the cell. ATIC is a bifunctional enzyme that catalyzes the final two steps of the de novo purine biosynthesis pathway (3Beardsley G.P. Rayl E.A. Gunn K. Moroson B.A. Seow H. Anderson K.S. Vergis J. Fleming K. Worland S. Condon B. Davies J. Adv. Exp. Med. Biol. 1998; 431: 221-226Crossref PubMed Scopus (28) Google Scholar). The AICAR transformylase domain (residues 199-592) catalyzes the transfer of the one-carbon formyl group from the co-factor N10-formyl-tetrahydrofolate (10-f-THF) to the substrate AICAR to produce 5-formyl-AICAR and tetrahydrofolate (see Fig. 1A). The cyclohydrolase domain (residues 1-198) then enhances the intramolecular cyclization of 5-formyl-AICAR to the final product of the pathway, IMP, via elimination of a water molecule. The individual AICAR transformylase and IMP cyclohydrolase domains can be expressed separately and are active (4Rayl E.A. Moroson B.A. Beardsley G.P. J. Biol. Chem. 1996; 271: 2225-2233Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Previous crystal structures of avian and human ATIC revealed an extensively intertwined homodimer (5Greasley S.E. Horton P. Ramcharan J. Beardsley G.P. Benkovic S.J. Wilson I.A. Nat. Struct. Biol. 2001; 8: 402-406Crossref PubMed Scopus (77) Google Scholar, 6Wolan D.W. Greasley S.E. Beardsley G.P. Wilson I.A. Biochemistry. 2002; 41: 15505-15513Crossref PubMed Scopus (37) Google Scholar, 7Wolan D.W. Greasley S.E. Wall M.J. Benkovic S.J. Wilson I.A. Biochemistry. 2003; 42: 10904-10914Crossref PubMed Scopus (22) Google Scholar) (see Fig. 1B) in which the transformylase and cyclohydrolase active sites were separated by ∼50 Å. However, no evidence of channeling or tunneling of 5-formyl-AICAR between the two domains has been forthcoming (5Greasley S.E. Horton P. Ramcharan J. Beardsley G.P. Benkovic S.J. Wilson I.A. Nat. Struct. Biol. 2001; 8: 402-406Crossref PubMed Scopus (77) Google Scholar, 8Bulock K.G. Beardsley G.P. Anderson K.S. J. Biol. Chem. 2002; 277: 22168-22174Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), although strong electropositive patches that connect the two active sites may sequester 5-formyl-AICAR to the vicinity of the binding sites. Cleland-type kinetic inhibition experiments indicated that the AICAR Tfase reaction is ordered and sequential with folate co-factor binding first to the AICAR Tfase active site (3Beardsley G.P. Rayl E.A. Gunn K. Moroson B.A. Seow H. Anderson K.S. Vergis J. Fleming K. Worland S. Condon B. Davies J. Adv. Exp. Med. Biol. 1998; 431: 221-226Crossref PubMed Scopus (28) Google Scholar). The formyl group is transferred directly from 10-f-THF to AICAR without any formylated enzyme-bound intermediate (9Smith G.K. Mueller W.T. Slieker L.J. DeBrosse C.W. Benkovic S.J. Biochemistry. 1982; 21: 2870-2874Crossref PubMed Scopus (30) Google Scholar), as evidenced from crystal structures of avian ATIC in complexes with AICAR (Protein Data Bank entry 1m9n) (6Wolan D.W. Greasley S.E. Beardsley G.P. Wilson I.A. Biochemistry. 2002; 41: 15505-15513Crossref PubMed Scopus (37) Google Scholar) and a multi-substrate adduct inhibitor (MSA) β-DADF (1o20) (7Wolan D.W. Greasley S.E. Wall M.J. Benkovic S.J. Wilson I.A. Biochemistry. 2003; 42: 10904-10914Crossref PubMed Scopus (22) Google Scholar). From these structures, His267 was proposed to increase the nucleophilicity of the 5-amino group of AICAR and then act as a catalytic base to deprotonate the AICAR amino group concomitant with nucleophilic attack of the substrate formyl group on 10-f-THF (6Wolan D.W. Greasley S.E. Beardsley G.P. Wilson I.A. Biochemistry. 2002; 41: 15505-15513Crossref PubMed Scopus (37) Google Scholar). Lys266 is then poised to stabilize the oxyanion transition state and shuttle protons to the N10 of tetrahydrofolate. The key role of Lys266 and His267 in this catalytic mechanism is further supported by kinetic studies of site-directed mutant enzymes (10Sugita T. Aya H. Ueno M. Ishizuka T. Kawashima K. J. Biochem. (Tokyo). 1997; 122: 309-313Crossref PubMed Scopus (18) Google Scholar, 11Shim J.H. Wall M. Benkovic S.J. Diaz N. Suarez D. Merz Jr., K.M. J. Am. Chem. Soc. 2001; 123: 4687-4696Crossref PubMed Scopus (16) Google Scholar), where mutation of Lys266 and His267 to alanine results in the complete loss of activity without significant change of the substrate Km (11Shim J.H. Wall M. Benkovic S.J. Diaz N. Suarez D. Merz Jr., K.M. J. Am. Chem. Soc. 2001; 123: 4687-4696Crossref PubMed Scopus (16) Google Scholar, 12Beardsley G.P. Rayl E.A. Gunn K. Moroson B.A. Seow H. Anderson K.S. Vergis J. Fleming K. Worland S. Condon B. Davies J. Griesmacher A. In Purine and Pyrimidine Metabolism in Man. Plenum Press, New York1997: 221-226Google Scholar). ATIC is highly conserved from Escherichia coli to human but has no sequence homology with other folate-dependent enzymes, such as GAR Tfase. Thus, folate-based inhibitors of GAR Tfase do not usually inhibit ATIC because of differential interactions within the two active sites (7Wolan D.W. Greasley S.E. Wall M.J. Benkovic S.J. Wilson I.A. Biochemistry. 2003; 42: 10904-10914Crossref PubMed Scopus (22) Google Scholar). For example, 6R-dideazatetrahydrofolate (Lometrexol), potently inhibits GAR Tfase (nm) but not ATIC (μm) (13Beardsley G.P. Moroson B.A. Taylor E.C. Moran R.G. J. Biol. Chem. 1989; 264: 328-333Abstract Full Text PDF PubMed Google Scholar, 14Erba E. Sen S. Sessa C. Vikhanskaya F.L. D'Incalci M. Br. J. Cancer. 1994; 69: 205-211Crossref PubMed Scopus (21) Google Scholar). Compared with the number of relatively potent inhibitors of GAR Tfase (15Boger D.L. Haynes N.E. Kitos P.A. Warren M.S. Ramcharan J. Marolewski A.E. Benkovic S.J. Bioorg. Med. Chem. 1997; 5: 1817-1830Crossref PubMed Scopus (42) Google Scholar, 16Boger D.L. Haynes N.E. Warren M.S. Ramcharan J. Kitos P.A. Benkovic S.J. Bioorg. Med. Chem. 1997; 5: 1853-1857Crossref PubMed Scopus (18) Google Scholar, 17Boger D.L. Haynes N.E. Warren M.S. Gooljarsingh L.T. Ramcharan J. Kitos P.A. Benkovic S.J. Bioorg. Med. Chem. 1997; 5: 1831-1838Crossref PubMed Scopus (25) Google Scholar, 19Boger D.L. Haynes N.E. Warren M.S. Ramcharan J. Marolewski A.E. Kitos P.A. Benkovic S.J. Bioorg. Med. Chem. 1997; 5: 1847-1852Crossref PubMed Scopus (19) Google Scholar, 20Boger D.L. Labroli M.A. Marsilje T.H. Jin Q. Hedrick M.P. Baker S.J. Shim J.H. Benkovic S.J. Bioorg. Med. Chem. 2000; 8: 1075-1086Crossref PubMed Scopus (17) Google Scholar, 21Zhang Y. Desharnais J. Marsilje T.H. Li C. Hedrick M.P. Gooljarsingh L.T. Tavassoli A. Benkovic S.J. Olson A.J. Boger D.L. Wilson I.A. Biochemistry. 2003; 42: 6043-6056Crossref PubMed Scopus (45) Google Scholar), specific inhibitors of ATIC have been scarce. However, Burroughs Wellcome (Research Triangle Park, NC) has designed and synthesized two antifolates that are specific (nm) for human ATIC, as compared with other folate-dependent enzymes, GAR Tfase, dihydrofolate reductase, and thymidylate synthase. These compounds are both sulfamido-bridged 5,8-dideazafolate analogs identified as BW2315U89UC (BW2315) and BW1540U88UD (BW1540) (see Fig. 1A) that differ only in the disposition of the imido and sulfonyl groups within the bridge region (see Fig. 1A). BW1540 and BW2315 have approximate Ki values against human ATIC of 8 and 6 nm, respectively, whereas the Ki values against GAR Tfase, dihydrofolate reductase and thymidylate synthase are within the micromolar range, except for BW1540, which showed low nanomolar inhibition against dihydrofolate reductase. 2R. Ferrone, personal communication. Cytotoxicity assays against human colon cell lines yielded an approximate IC50 of 0.7-3 μm for BW1540 and 1-5 μm for BW2315. To elucidate their mechanism of inhibition, BW1540 and BW2315 were co-crystallized with human ATIC in the presence of substrate AICAR. These first human ATIC ternary complexes with independently bound substrate and folate moieties not only advance the ATIC mechanistic studies but provide insights into the future design of antifolates selective against ATIC. Materials—Luria broth and agar were obtained from Invitrogen. All common buffers and reagents were purchased from Sigma-Aldrich. The folate-based inhibitors BW1540U88UD and BW2315U89UC were kind gifts from Dr. Robert Ferrone (Burroughs Wellcome). Protein Preparation and Purification—Human ATIC was prepared as previously reported (22Wolan D.W. Cheong C.G. Greasley S.E. Wilson I.A. Biochemistry. 2004; 43: 1171-1183Crossref PubMed Scopus (30) Google Scholar). Inhibitors BW1540 and BW2315 and substrate AICAR were added in a 10-fold molar excess to human ATIC protein (0.1-0.4 mg/ml), heated in a 37 °C water bath for 30 min to prevent precipitation, and then incubated overnight at 4 °C. The protein solution was concentrated to 10 mg/ml using Millipore Ultrafree-15 filters (molecular mass of 10,000 Da) and stored at 4 °C for crystallization experiments. Crystallization and Data Collection—Crystals of human ATIC in complex with AICAR and BW1540 were grown at 4 °C by sitting drop vapor diffusion by mixing equal volumes of human ATIC (10 mg/ml) and a reservoir solution consisting of 17% polyethylene glycol 3000, 0.1 m Tris, pH 7.5, 10% glycerol, 6 mm dithiothreitol, and 0.1 m NaCl. Streak seeding (23Stura E.A. Wilson I.A. J. Cryst. Growth. 1991; 110: 270-282Crossref Scopus (142) Google Scholar) with apo human ATIC crystals that were produced in similar conditions facilitated growth of needle-shaped crystals. The data were collected to 2.55 Å resolution on a single, flash-cooled crystal at 83 K in a cryoprotectant consisting of mother liquor and 20% glycerol on Beamline 11.1 at the Stanford Synchrotron Radiation Laboratory (Menlo Park, CA). The data were processed to 2.55 Å with HKL2000 (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar) in orthorhombic space group P212121 (a = 83.60 Å, b = 93.04 Å, and c = 164.19 Å) (Table I). The calculated Matthews' coefficient (VM = 2.5 Å3 Da-1) suggested two monomers per asymmetric unit (∼50% solvent). Crystals of human ATIC in complex with AICAR and BW2315 were similarly produced as described above. The data were collected to 2.6 Å resolution on a single flash-cooled crystal at 106 K on Beamline 5.0.2 at Advanced Light Source (Berkeley, CA). The data were processed with HKL2000 (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar) in monoclinic space group P21 (a = 77.12 Å, b = 92.97 Å, c = 178.49 Å, and β = 91.19°) (Table I). The calculated Matthews' coefficient (VM = 2.5 Å3 Da-1) suggested four monomers/asymmetric unit (∼50% solvent).Table IData collection statisticsBW1540U88UD complexBW2315U89UC complexSpace groupP212121P21Unit cell dimensions (Å; degree)a = 83.60, b = 93.04, c = 164.19a = 77.12, b = 92.97, c = 178.49, β = 91.2Molecules/AU24Resolution range (Å)aNumbers in parentheses refer to the highest resolution shell.30-2.55 (2.64-2.55)20-2.60 (2.64-2.60)Average I/σ13.2 (2.9)8.7 (1.5)Unique reflections42,466 (4,156)71,039 (3,597)Redundancy3.8 (3.8)2.7 (2.4)Completeness (%)100.0 (100.0)91.3 (92.3)Rsym (%)bRsym=Σ[|Ihi|-|Ih|]/ΣIhi×100, where Ihi and Ih are the intensities of individual and mean structure factors, respectively.11.0 (48.9)12.4 (65.9)a Numbers in parentheses refer to the highest resolution shell.b Rsym=Σ[|Ihi|-|Ih|]/ΣIhi×100, where Ihi and Ih are the intensities of individual and mean structure factors, respectively. Open table in a new tab Structure Solution and Refinement—The BW1540 and BW2315 complex structures were both determined by molecular replacement using the apo avian ATIC structure (1g8m) (5Greasley S.E. Horton P. Ramcharan J. Beardsley G.P. Benkovic S.J. Wilson I.A. Nat. Struct. Biol. 2001; 8: 402-406Crossref PubMed Scopus (77) Google Scholar) as the initial search model. Molecular replacement was performed with EPMR (25Kissinger C.R. Gehlhaar D.K. Fogel D.B. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 484-491Crossref PubMed Scopus (691) Google Scholar) for the BW1540 structure solution, whereas AMoRe (26Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) was used for the BW2315 complex. For the BW1540 complex, the rotation and translation search, using all data from 15.0-3.5 Å, identified two monomers in the asymmetric unit with a final correlation co-efficient and Rcryst of 55.5 and 44.3%, respectively (note that for the first noise peak, correlation coefficient = 26.4 and Rcryst = 56.2%). The two monomers form the biologically relevant homodimer as observed in previous avian ATIC structures (5Greasley S.E. Horton P. Ramcharan J. Beardsley G.P. Benkovic S.J. Wilson I.A. Nat. Struct. Biol. 2001; 8: 402-406Crossref PubMed Scopus (77) Google Scholar, 6Wolan D.W. Greasley S.E. Beardsley G.P. Wilson I.A. Biochemistry. 2002; 41: 15505-15513Crossref PubMed Scopus (37) Google Scholar, 7Wolan D.W. Greasley S.E. Wall M.J. Benkovic S.J. Wilson I.A. Biochemistry. 2003; 42: 10904-10914Crossref PubMed Scopus (22) Google Scholar). For the BW2315 complex, the rotation search (15.0-4.0 Å) identified four major solutions with correlation co-efficients of 13.9-12.1 (noise peak = 8.8); the subsequent translational search yielded a final correlation co-efficient of 60.8 and a Rcryst of 39.4%. The four monomers form two biologically relevant homodimers. Both models were manually built with O (27Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) and TURBO-FRODO (28Roussel A. Cambillau C. Silicon Graphics Geometry Partners Directory.Vol. 86. Silicon Graphics, Mountain View, CA1991Google Scholar) and refined using CNS (29Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) with cycles of conventional positional refinement and simulated annealing (MLF target). Difference electron density maps clearly identified inhibitors BW1540 and BW2315, as well as an endogenously bound xanthosine 5′-monophosphate (XMP), within the AICAR Tfase and IMP cyclohydrolase active sites, respectively. Approximate 2-fold noncrystallographic symmetry restraints were applied during the initial round of refinements and then released later to accommodate differences within the individual monomers. Water molecules were automatically positioned by CNS using a 3 σ cut-off in Fo-Fc maps and manually inspected. For the BW1540 structure, the final Rcryst and Rfree are 18.9 and 25.7%, respectively. For the BW2315 structure, the final Rcryst and Rfree are 21.3 and 27.5%, respectively (Table II).Table IIRefinement statisticsBW1540 U88UD complexBW2315U89UC complexResolution (Å)2.552.60Rcryst/Rfree (%)aRcryst=(Σ|Fo|-k|Fc|/Σ|Fo|)×100 where Fo and Fc are the observed and calculated structure f actors, respectively. Rfree is computed as described for Rcryst, but with the test set of reflections only, reflections only.18.9/25.721.3/27.5Reflections used (test set)37,931 (1,986)64,075 (3,426)Protein atoms8,93317,397Water molecules399388Ions, ligands2 potassium ions4 potassium ions2 AICAR3 AICAR2 BW1540U88UD2 BW23 15U89UC1 XMP2 XMPB values (Å2)Protein30.126.8Water28.724.5Ligands31.727.9Root mean square deviationsBond length (Å)0.0080.008Bond angle (degree)1.401.39Coordinate errorbCross-validated Luzzati coordinate error.0.39 Å0.42 ÅRamachandran plot (%)Most favored88.688.0Additional11.111.6Generous0.10.4Disallowed0.20.0Protein Data Bank code1p4r1p10a Rcryst=(Σ|Fo|-k|Fc|/Σ|Fo|)×100 where Fo and Fc are the observed and calculated structure f actors, respectively. Rfree is computed as described for Rcryst, but with the test set of reflections only, reflections only.b Cross-validated Luzzati coordinate error. Open table in a new tab The models were analyzed with PROCHECK (30Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), WHATCHECK (31Hooft R.W. Vriend G. Sander C. Abola E.E. Nature. 1996; 381: 272Crossref PubMed Scopus (1818) Google Scholar), CNS (29Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar), and CCP4 (32CCP4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). Sc co-efficients and buried surface areas were calculated with SC (32CCP4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) and MS (33Connolly M.L. Science. 1983; 221: 709-713Crossref PubMed Scopus (2453) Google Scholar) using 1.7 and 1.4 Å probes, respectively. Analysis with PROCHECK (30Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) revealed that 88.6 and 88.0% of the residues are located in the most favorable regions in the Ramachandran plot for the BW1540 and BW2315 complexes, respectively. In both structures, a surface Asp502 lies in the disallowed region of the Ramachandran plot, located near the generously allowed region, but has well ordered electron density. Figs. 1B, 2, 3, 4, 5, 6 were created with PyMol (34DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). The coordinates and structure factors have been deposited in the Protein Data Bank (35Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27935) Google Scholar) with accession entries 1p4r (BW1540U88UD) and 1pl0 (BW2315U89UC).Fig. 3The AICAR Tfase active site with BW antifolates bound. A, electrostatic and hydrogen bonding residues in the AICAR Tfase active site that interact with antifolate BW1540. BW1540 and ATIC residues are represented in ball-and-stick with BW1540 atoms colored as in Fig. 1B. Carbon atoms for the active site residues are colored blue for subunit A and purple for subunit B of the dimer with structural water molecules represented as red spheres. B, corresponding view of hydrogen bonding residues within the AICAR Tfase active site that interact with AICAR and antifolate BW2315. BW2315 and ATIC residues are represented in ball-and-stick with BW2315 atoms colored as in Fig. 1B. Carbon atoms for the active site residues are colored blue for subunit C and purple for subunit D with structural water molecules represented as red spheres. C, stereo view of the AICAR Tfase active site bound with BW1540 depicts the hydrophobic residues within the folate-binding region. BW1540 and ATIC residues are labeled and colored according to A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Comparison of the interactions of the AICAR Tfase active site with BW1540 and BW2315. A, schematic representation of the hydrogen bonding network and corresponding distances within the active site between AICAR, BW1540 and the AICAR Tfase residues. Interacting residues from the opposite subunit of the AICAR-bound monomer (black) are labeled in blue and indicated with a prime symbol. B, schematic representation of the hydrogen bonding network and corresponding distances within the AICAR Tfase active site with the bound AICAR and BW2315 molecules. Interacting residues from the opposite subunit of the AICAR-bound monomer (black) are labeled in blue and are indicated with a prime symbol.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Comparison of free and bound AICAR Tfase active sites. A, superposition of the AICAR- and antifolate-bound human AICAR Tfase active sites reveals slight translational and orientational deviations between the inhibitor molecules because of the propensity for the sulfonyl groups to be located in the oxyanion hole. The BW1540-bound structure is colored and labeled according to Fig. 3A. BW2315 carbons are colored orange with the BW2315-bound ATIC carbons and Cα trace colored in wheat. B, superposition of the BW1540-bound structure and the apo AICAR Tfase active sites reveals the slight conformational changes that occur upon folate binding. The BW1540-bound structure is colored and labeled according to Fig. 3A with the apo human ATIC Cα trace colored in wheat.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6BW1540, BW2315,and β-DADFand active site catalytic residue superposition for all known ATIC structures. A, two rotated views of the overlay of β-DADF and the BW antifolates reveals slight variations in the positioning of the MSA because of the trans-double bond linker between the AICAR and folate moieties. The BW1540-bound human structure is colored as in Fig. 1B, with the BW2315-bound ATIC carbons colored teal and the β-DADF-bound carbons colored salmon. B, determination of the antifolate-bound human ATIC structures reveals Asn431′ positions both the AICAR 5-amino and 10-formyl group of folate for optimal transformylation. Previous structures show Asn431′ pointing away from the bound substrates with an active site water molecule positioned near the 5-amino group of AICAR. The AICAR and BW1540 inhibitor of the BW1540-bound human structure are colored as in Fig. 1B with Lys266, His267 and Ans431′ of the human BW1540-bound (cyan carbons, 1p4r), apo human (forest green carbons, 1pxk), avian AICAR/XMP-bound (wheat carbons, 1m9n), and avian β-DADF-bound (grey carbons, 1o20) depicted.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The crystal structures of human ATIC in complex with antifolates BW1540U88UD and BW2315U89UC provide the first view of how the substrate AICAR and folate-based co-factor moieties simultaneously occupy the AICAR Tfase active site. The AICAR Tfase active site is located in a long, narrow cleft at the dimer interface where AICAR interacts primarily with one subunit and folate with the opposing subunit (7Wolan D.W. Greasley S.E. Wall M.J. Benkovic S.J. Wilson I.A. Biochemistry. 2003; 42: 10904-10914Crossref PubMed Scopus (22) Google Scholar). The refined human ATIC BW1540 structure consists of residues 4-592 in monomer A and residues 5-592 in monomer B with 399 water molecules, two potassium ions, two BW1540 inhibitors within the AICAR Tfase domains, but only one XMP in the IMP cyclohydrolase active site of monomer A (Fig. 1B). XMP is carried through protein purification and crystallization and also selectively binds to only one of the two IMP cyclohydrolase active sites in the homodimer in apo avian and human ATIC structures (5Greasley S.E. Horton P. Ramcharan J. Beardsley G.P. Benkovic S.J. Wilson I.A. Nat. Struct. Biol. 2001; 8: 402-406Crossref PubMed Scopus (77) Google Scholar, 22Wolan D.W. Cheong C.G. Greasley S.E. Wilson I.A. Biochemistry. 2004; 43: 1171-1183Crossref PubMed Scopus (30) Google Scholar, 36Vergis J. Beardsley G.P. Biochemistry. 2004; 43: 1184-1192Crossref PubMed Scopus (17) Google Scholar). The final model for the BW2315 complex consists of two dimers (i.e. monomers A (4-592) and B (4-592) compose one dimer, and monomers C (5-592) and D (4-592) compose the second dimer) and 388 water molecules. No main chain density was observed between residues 482 and 485 (B), 481-485 (C), and 477-486 (D). Each monomer has a bound potassium ion within the AICAR Tfase domain with only one XMP per dimer (monomers A and C). Interpretable density for the substrate AICAR was found only in monomers B, C, and D, whereas interpretable BW2315 density was found only in monomers B and D. The AICAR Tfase domain contains three subdomains (domains 2-4) as previously described (5Greasley S.E. Horton P. Ramcharan J. Beardsley G.P. Benkovic S.J. Wilson I.A. Nat. Struct. Biol. 2001; 8: 402-406Crossref PubMed Scopus (77) Google Scholar, 6Wolan D.W. Greasley S.E. Beardsley G.P. Wilson I.A. Biochemistry. 2002; 41: 15505-15513Crossref PubMed Scopus (37) Google Scholar, 7Wolan D.W. Greasley S.E. Wall M.J. Benkovic S.J. Wilson I.A. Biochemistry. 2003; 42: 10904-10914Crossref PubMed Scopus (22) Google Scholar). For each complex, domain 3 has higher B values (41-47 Å2) compared with the overall protein B values (27-30 Å2) as previously found (6Wolan D.W. Greasley S.E. Beardsley G.P. Wilson I.A. Biochemistry. 2002; 41: 15505-15513Crossref PubMed Scopus (37) Google Scholar, 7Wolan D.W. Greasley S.E. Wall M.J. Benkovic S.J. Wilson I.A. Biochemistry. 2003; 42: 10904-10914Crossref PubMed Scopus (22) Google Scholar). AICAR Interactions for Both the BW1540- and BW2315-bound Human ATIC Structures—Clear Fo-Fc electron density for the AICAR substrate was present in the AICAR Tfase active sites of both complexes, except for monomer A of the BW2315-bound complex structure, which had weak density that could not be unequivocally interpreted. AICAR binds to both complex structures in a similar conformation with a C3′-endo sugar pucker (Fig. 2). The 4-carboxamide ami

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