Multiple Conformers in Active Site of Human Dihydrofolate Reductase F31R/Q35E Double Mutant Suggest Structural Basis for Methotrexate Resistance
2009; Elsevier BV; Volume: 284; Issue: 30 Linguagem: Inglês
10.1074/jbc.m109.018010
ISSN1083-351X
AutoresJordan P. Volpato, Brahm J. Yachnin, Jonathan Blanchet, Vanessa Guerrero, Lucie Poulin, Elena Fossati, Albert M. Berghuis, Joelle N. Pelletier,
Tópico(s)HIV/AIDS drug development and treatment
ResumoMethotrexate is a slow, tight-binding, competitive inhibitor of human dihydrofolate reductase (hDHFR), an enzyme that provides key metabolites for nucleotide biosynthesis. In an effort to better characterize ligand binding in drug resistance, we have previously engineered hDHFR variant F31R/Q35E. This variant displays a >650-fold decrease in methotrexate affinity, while maintaining catalytic activity comparable to the native enzyme. To elucidate the molecular basis of decreased methotrexate affinity in the doubly substituted variant, we determined kinetic and inhibitory parameters for the simple variants F31R and Q35E. This demonstrated that the important decrease of methotrexate affinity in variant F31R/Q35E is a result of synergistic effects of the combined substitutions. To better understand the structural cause of this synergy, we obtained the crystal structure of hDHFR variant F31R/Q35E complexed with methotrexate at 1.7-Å resolution. The mutated residue Arg-31 was observed in multiple conformers. In addition, seven native active-site residues were observed in more than one conformation, which is not characteristic of the wild-type enzyme. This suggests that increased residue disorder underlies the observed methotrexate resistance. We observe a considerable loss of van der Waals and polar contacts with the p-aminobenzoic acid and glutamate moieties. The multiple conformers of Arg-31 further suggest that the amino acid substitutions may decrease the isomerization step required for tight binding of methotrexate. Molecular docking with folate corroborates this hypothesis. Methotrexate is a slow, tight-binding, competitive inhibitor of human dihydrofolate reductase (hDHFR), an enzyme that provides key metabolites for nucleotide biosynthesis. In an effort to better characterize ligand binding in drug resistance, we have previously engineered hDHFR variant F31R/Q35E. This variant displays a >650-fold decrease in methotrexate affinity, while maintaining catalytic activity comparable to the native enzyme. To elucidate the molecular basis of decreased methotrexate affinity in the doubly substituted variant, we determined kinetic and inhibitory parameters for the simple variants F31R and Q35E. This demonstrated that the important decrease of methotrexate affinity in variant F31R/Q35E is a result of synergistic effects of the combined substitutions. To better understand the structural cause of this synergy, we obtained the crystal structure of hDHFR variant F31R/Q35E complexed with methotrexate at 1.7-Å resolution. The mutated residue Arg-31 was observed in multiple conformers. In addition, seven native active-site residues were observed in more than one conformation, which is not characteristic of the wild-type enzyme. This suggests that increased residue disorder underlies the observed methotrexate resistance. We observe a considerable loss of van der Waals and polar contacts with the p-aminobenzoic acid and glutamate moieties. The multiple conformers of Arg-31 further suggest that the amino acid substitutions may decrease the isomerization step required for tight binding of methotrexate. Molecular docking with folate corroborates this hypothesis. Human dihydrofolate reductase (hDHFR) 6The abbreviations used are: hDHFRhuman dihydrofolate reductaseMES4-morpholineethanesulfonic acidDHFdihydrofolateMTXmethotrexateMTXON-[4-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]methylamino]benzoyl]-l-glutamatep-ABAp-aminobenzoic acid. 6The abbreviations used are: hDHFRhuman dihydrofolate reductaseMES4-morpholineethanesulfonic acidDHFdihydrofolateMTXmethotrexateMTXON-[4-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]methylamino]benzoyl]-l-glutamatep-ABAp-aminobenzoic acid. catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate in a NADPH-dependent manner. 5,6,7,8-Tetrahydrofolate is a cofactor in purine and thymidylate biosynthesis, which are essential metabolites in cell division and proliferation. As a consequence of its essential role in nucleoside biosynthesis, hDHFR has been extensively exploited as a drug target. Inhibition with folate antagonists, or antifolates, arrests cell proliferation. The most effective clinical antifolate to date is methotrexate (MTX (Fig. 1)), a slow, tight-binding competitive inhibitor that displays high affinity for hDHFR (KiMTX = 3.4 pm). MTX is currently used to treat a variety of diseases, including cancer (1Slamon D.J. Romond E.H. Perez E.A. Clin. Adv. Hematol. Oncol. 2006; 4: 4-9Google Scholar, 2Daw N.C. Billups C.A. Rodriguez-Galindo C. McCarville M.B. Rao B.N. Cain A.M. Jenkins J.J. Neel M.D. Meyer W.H. Cancer. 2006; 106: 403-412Crossref PubMed Scopus (96) Google Scholar, 3Strojan P. Soba E. Budihna M. Auersperg M. J. Surg. Oncol. 2005; 92: 278-283Crossref PubMed Scopus (27) Google Scholar), and autoimmune diseases such as juvenile idiopathic arthritis (4Ramanan A.V. Whitworth P. Baildam E.M. Arch. Dis. Child. 2003; 88: 197-200Crossref PubMed Scopus (97) Google Scholar). A number of resistance mechanisms to MTX have been observed in cancer patients, including impaired transport of MTX to the cytoplasm (5Flintoff W.F. Sadlish H. Gorlick R. Yang R. Williams F.M. Biochim. Biophys. Acta. 2004; 1690: 110-117Crossref PubMed Scopus (24) Google Scholar) and decreased retention of MTX in the cell (6Takemura Y. Kobayashi H. Miyachi H. Anticancer Drugs. 1999; 10: 677-683Crossref PubMed Scopus (9) Google Scholar). Numerous ex vivo studies have reported mutations in the hDHFR gene resulting in an enzyme variant with decreased affinity for MTX (7Blakley R.L. Sorrentino B.P. Hum. Mutat. 1998; 11: 259-263Crossref PubMed Scopus (31) Google Scholar, 8Volpato J.P. Pelletier J.N. Drug Resistance Updates. 2009; 12: 28-41Crossref PubMed Scopus (31) Google Scholar). These have contributed to increase our understanding of the molecular basis for active-site discrimination between the substrate, DHF, and its competitive inhibitor, MTX. Understanding the molecular interactions that affect tight binding of MTX to the active site of DHFR will contribute to our understanding of antifolate binding to DHFR, which can in turn contribute to the design of more efficient inhibitors. human dihydrofolate reductase 4-morpholineethanesulfonic acid dihydrofolate methotrexate N-[4-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]methylamino]benzoyl]-l-glutamate p-aminobenzoic acid. human dihydrofolate reductase 4-morpholineethanesulfonic acid dihydrofolate methotrexate N-[4-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]methylamino]benzoyl]-l-glutamate p-aminobenzoic acid. A considerable number of DHFR active-site variants have been identified in MTX-resistant cancer cell lines (although never in patients) (9Spencer H.T. Sorrentino B.P. Pui C.H. Chunduru S.K. Sleep S.E. Blakley R.L. Leukemia. 1996; 10: 439-446PubMed Google Scholar) or engineered in vitro to elucidate the role of active site residues in the binding of MTX. Amino acid substitutions at residues Ile-7 (10Patel M. Sleep S.E. Lewis W.S. Spencer H.T. Mareya S.M. Sorrentino B.P. Blakley R.L. Hum. Gene Ther. 1997; 8: 2069-2077Crossref PubMed Scopus (27) Google Scholar), Leu-22 (11Ercikan-Abali E.A. Waltham M.C. Dicker A.P. Schweitzer B.I. Gritsman H. Banerjee D. Bertino J.R. Mol. Pharmacol. 1996; 49: 430-437PubMed Google Scholar, 12Lewis W.S. Cody V. Galitsky N. Luft J.R. Pangborn W. Chunduru S.K. Spencer H.T. Appleman J.R. Blakley R.L. J. Biol. Chem. 1995; 270: 5057-5064Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), Phe-31 (13Chunduru S.K. Cody V. Luft J.R. Pangborn W. Appleman J.R. Blakley R.L. J. Biol. Chem. 1994; 269: 9547-9555Abstract Full Text PDF PubMed Google Scholar), Phe-34 (14Nakano T. Spencer H.T. Appleman J.R. Blakley R.L. Biochemistry. 1994; 33: 9945-9952Crossref PubMed Scopus (31) Google Scholar), Arg-70 (15Thompson P.D. Freisheim J.H. Biochemistry. 1991; 30: 8124-8130Crossref PubMed Scopus (21) Google Scholar), and Val-115 (16Fossati E. Volpato J.P. Poulin L. Guerrero V. Dugas D.A. Pelletier J.N. J. Biomol. Screen. 2008; 13: 504-514Crossref PubMed Scopus (13) Google Scholar) have yielded MTX-resistant variants. These residues are all present in the folate-binding pocket (17Cody V. Luft J.R. Pangborn W. Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 147-155Crossref PubMed Scopus (86) Google Scholar). Because MTX and DHF bind to the active site of hDHFR in a similar manner, all known substitutions causing a decrease in MTX affinity also decrease DHF affinity and overall catalytic efficiency (7Blakley R.L. Sorrentino B.P. Hum. Mutat. 1998; 11: 259-263Crossref PubMed Scopus (31) Google Scholar, 16Fossati E. Volpato J.P. Poulin L. Guerrero V. Dugas D.A. Pelletier J.N. J. Biomol. Screen. 2008; 13: 504-514Crossref PubMed Scopus (13) Google Scholar, 18Volpato J.P. Fossati E. Pelletier J.N. J. Mol. Biol. 2007; 373: 599-611Crossref PubMed Scopus (31) Google Scholar). However, the loss of DHF affinity and catalytic efficiency is generally smaller than the loss of MTX affinity. This is often attributed to formation of different contacts with either ligand due to the 180° inversion of the pterin ring of bound DHF relative to MTX (17Cody V. Luft J.R. Pangborn W. Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 147-155Crossref PubMed Scopus (86) Google Scholar, 19Oefner C. D'Arcy A. Winkler F.K. Eur. J. Biochem. 1988; 174: 377-385Crossref PubMed Scopus (196) Google Scholar). Crystal structures of MTX-resistant point mutants have offered insight into the causes of decreased binding of MTX or other antifolates (17Cody V. Luft J.R. Pangborn W. Acta Crystallogr. D Biol. Crystallogr. 2005; 61: 147-155Crossref PubMed Scopus (86) Google Scholar, 20Cody V. Luft J.R. Pangborn W. Gangjee A. Queener S.F. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 646-655Crossref PubMed Scopus (29) Google Scholar, 21Cody V. Luft J.R. Pangborn W. Gangjee A. Acta Crystallogr. D Biol. Crystallogr. 2003; 59: 1603-1609Crossref PubMed Scopus (19) Google Scholar, 22Cody V. Galitsky N. Luft J.R. Pangborn W. Gangjee A. Acta Crystallogr. D. Biol. Crystallogr. 2003; 59: 654-661Crossref PubMed Scopus (12) Google Scholar, 23Gangjee A. Vidwans A.P. Vasudevan A. Queener S.F. Kisliuk R.L. Cody V. Li R. Galitsky N. Luft J.R. Pangborn W. J. Med. Chem. 1998; 41: 3426-3434Crossref PubMed Scopus (64) Google Scholar, 24Cody V. Galitsky N. Luft J.R. Pangborn W. Rosowsky A. Blakley R.L. Biochemistry. 1997; 36: 13897-13903Crossref PubMed Scopus (58) Google Scholar). To this day, crystal structures of MTX-resistant hDHFR variants L22F, L22R, and L22Y (12Lewis W.S. Cody V. Galitsky N. Luft J.R. Pangborn W. Chunduru S.K. Spencer H.T. Appleman J.R. Blakley R.L. J. Biol. Chem. 1995; 270: 5057-5064Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), as well as F31G and F31S (25Cody V. Galitsky N. Luft J.R. Pangborn W. Blakley R.L. Gangjee A. Anticancer Drug Des. 1998; 13: 307-315PubMed Google Scholar), complexed to various antifolates, have been reported. Only the L22Y variant has been co-crystallized with MTX. Despite its decreased affinity for MTX (L22Y KiMTX = 11 nm versus WT KiMTX < 31 pm (18Volpato J.P. Fossati E. Pelletier J.N. J. Mol. Biol. 2007; 373: 599-611Crossref PubMed Scopus (31) Google Scholar)), the inhibitor in the variant structure was bound in the same way as in the native enzyme, making interpretation of decreased affinity difficult to assess. Nonetheless, the low probability conformation of residue Tyr-22 suggested that the presence of a bulky aromatic residue in this area of the folate-binding pocket generated unfavorable hydrophobic interactions with the 2,4-diaminopterin moiety of the inhibitor (12Lewis W.S. Cody V. Galitsky N. Luft J.R. Pangborn W. Chunduru S.K. Spencer H.T. Appleman J.R. Blakley R.L. J. Biol. Chem. 1995; 270: 5057-5064Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). This is also expected to reduce DHF substrate binding. Structures of MTX-resistant variants F31G and F31S were obtained complexed to N-[4-[(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]methylamino]benzoyl]-l-glutamate (MTXO) (25Cody V. Galitsky N. Luft J.R. Pangborn W. Blakley R.L. Gangjee A. Anticancer Drug Des. 1998; 13: 307-315PubMed Google Scholar), a MTX analog in which the 2,4–2,4-diaminopterin moiety is replaced by a 2,4-diaminofuropyrimidine moiety. Superposition of MTXO-bound variants with MTX-bound WT hDHFR revealed that the ligands bind to the active site in an analogous manner. It was suggested that decreased MTX binding in the substituted variants resulted from the loss of van der Waals and hydrophobic contacts established between the native Phe-31 and the p-ABA and 2,4-diaminopterin moieties of MTX. F31G and F31S display a 10-fold decrease in affinity for MTX relative to WT hDHFR (KiMTX < 31 pm (18Volpato J.P. Fossati E. Pelletier J.N. J. Mol. Biol. 2007; 373: 599-611Crossref PubMed Scopus (31) Google Scholar)). Further Phe-31 variants (i.e. F31R; KiMTX = 7 nm, 200-fold decrease in MTX affinity) (10Patel M. Sleep S.E. Lewis W.S. Spencer H.T. Mareya S.M. Sorrentino B.P. Blakley R.L. Hum. Gene Ther. 1997; 8: 2069-2077Crossref PubMed Scopus (27) Google Scholar) display larger decreases in affinity relative to F31G and F31S. This cannot be rationalized by reduction of side-chain contacts with the inhibitor due to the presence of a smaller side chain. These results illustrate the difficulty of gaining insight into the molecular causes for altered MTX binding. This may be partly attributed to the very tight binding of MTX to the native enzyme, such that binding to resistant variants often remains in the sub-nanomolar or low nanomolar range, where the general mode of ligand binding has not changed appreciably relative to the native enzyme. Combining active-site mutations in hDHFR by protein engineering has been shown to generate variants with greatly decreased affinity to MTX (18Volpato J.P. Fossati E. Pelletier J.N. J. Mol. Biol. 2007; 373: 599-611Crossref PubMed Scopus (31) Google Scholar, 26Ercikan-Abali E.A. Mineishi S. Tong Y. Nakahara S. Waltham M.C. Banerjee D. Chen W. Sadelain M. Bertino J.R. Cancer Res. 1996; 56: 4142-4145PubMed Google Scholar). Studying the molecular interactions in highly MTX-resistant hDHFR variants offers the possibility of capturing more important changes in enzyme-ligand interactions. Here, we report detailed observations for the mode of MTX resistance in the combinatorial variant F31R/Q35E. Variant F31R/Q35E is a relevant candidate for better understanding the specific interactions that govern ligand recognition in the folate binding site, because it displays a >650-fold decrease in MTX affinity (KiMTX = 21 nm) accompanied by a modest, 9-fold decrease of affinity for the substrate DHF relative to WT hDHFR (18Volpato J.P. Fossati E. Pelletier J.N. J. Mol. Biol. 2007; 373: 599-611Crossref PubMed Scopus (31) Google Scholar). In addition, we have recently shown that this variant is an efficient selectable marker for various mammalian cell types, including murine hematopoietic stem cells (18Volpato J.P. Fossati E. Pelletier J.N. J. Mol. Biol. 2007; 373: 599-611Crossref PubMed Scopus (31) Google Scholar). 7J. P. Volpato, N. Mayotte, G. Sauvageau, and J. N. Pelletier, submitted for publication. 7J. P. Volpato, N. Mayotte, G. Sauvageau, and J. N. Pelletier, submitted for publication. Because mutations giving rise to MTX resistance are not observed in mammals, and because MTX is approved for human treatment, engineered resistant DHFRs offer great potential as human selective markers ex vivo or in vivo (10Patel M. Sleep S.E. Lewis W.S. Spencer H.T. Mareya S.M. Sorrentino B.P. Blakley R.L. Hum. Gene Ther. 1997; 8: 2069-2077Crossref PubMed Scopus (27) Google Scholar, 27Allay J.A. Persons D.A. Galipeau J. Riberdy J.M. Ashmun R.A. Blakley R.L. Sorrentino B.P. Nat. Med. 1998; 4: 1136-1143Crossref PubMed Scopus (180) Google Scholar, 28Flasshove M. Banerjee D. Mineishi S. Li M.X. Bertino J.R. Moore M.A. Blood. 1995; 85: 566-574Crossref PubMed Google Scholar). To better understand the effect of either amino acid substitution on each ligand, a kinetic double mutant cycle was constructed with the simple variants F31R and Q35E. The crystal structure of the F31R/Q35E variant was obtained with bound MTX at 1.7-Å resolution, to elucidate the structural basis of MTX resistance in this variant. In addition, molecular docking was performed with the F31R/Q35E structure to evaluate the role of the two substitutions toward folate binding. Overall, the results reveal synergistic effects of the combined substitutions toward loss of MTX binding, characterized by increased disorder of specific residues throughout the active site of the highly MTX-resistant F31R/Q35E variant. The hDHFR F31R/Q35E gene was amplified by PCR using the following primer set: forward (5′-CACACACCATATGGTTGGTTCGCTAAACTG-3′, NdeI restriction site in italics) and reverse (5′-GTTCTGAGGTCATTACTGG-3′, external primer) from the hDHFR F31R/Q35E-pQE32 template (18Volpato J.P. Fossati E. Pelletier J.N. J. Mol. Biol. 2007; 373: 599-611Crossref PubMed Scopus (31) Google Scholar). The amplified gene was subcloned in the modified pET24 vector (29Doucet N. Savard P.Y. Pelletier J.N. Gagné S.M. J. Biol. Chem. 2007; 282: 21448-21459Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) between the NdeI and HindIII restriction sites using T4 DNA ligase, and the ligation mixture was transformed into electrocompetent BL21(DE3) cells. The expected sequence was confirmed by DNA sequencing. The F31R and Q35E substitutions were created by megaprimer PCR using external primer set 2 described in a previous study (18Volpato J.P. Fossati E. Pelletier J.N. J. Mol. Biol. 2007; 373: 599-611Crossref PubMed Scopus (31) Google Scholar) and the mutagenic primers 5′-TCTCTGGAAATATCTACGTTCGTTCCTTAAGG (F31R, reverse) and 5′-GTTGTGGTCATTTCTTTCGAAATATCTAAATTCGT (Q35E, reverse), respectively. The amplified gene was subcloned in the pQE32 vector between the BamHI and HindIII restriction sites using T4 DNA ligase, and the ligation mixture was transformed into electrocompetent SK037 cells (18Volpato J.P. Fossati E. Pelletier J.N. J. Mol. Biol. 2007; 373: 599-611Crossref PubMed Scopus (31) Google Scholar). Expression, purification, and determination of kinetic and inhibitory constants was performed as previously described (18Volpato J.P. Fossati E. Pelletier J.N. J. Mol. Biol. 2007; 373: 599-611Crossref PubMed Scopus (31) Google Scholar). Briefly, kinetic and inhibition assays were conducted in MATS buffer (25 mm MES, 25 mm acetate, 50 mm Tris, 100 mm sodium acetate, and 0.02% (w/v) sodium azide) (pH 7.6) at 23 °C, by monitoring the NADPH and DHF depletion (Δϵ340 nm = 12 800 m−1 cm−1). All assays were performed in at least four independent experiments, and the average values are reported. The initial rates during the first 15% of substrate conversion were recorded for all assays. Kinetic and inhibition parameters were obtained from a non-linear regression fit to the Henri Michaelis-Menten equation using Prism (GraphPad Software, San Diego, CA). The kcat values were determined in the presence of saturating substrate concentrations (100 μm each of DHF and NADPH) in 1-cm cells according to kcat = Vmax/[E]. KmDHF values were obtained using 10-cm cells containing 1 nm enzyme, 10 μm NADPH and a range of DHF concentrations (0.05 μm to 10 μm). IC50MTX was determined in the presence of saturating substrate concentrations and variable MTX concentrations (0.025 μm to 100 μm). Inhibition constants for MTX (KiMTX) were calculated from IC50MTX according to the equation for competitive inhibitor binding (30Segel I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems. Wiley Classics Library Ed John Wiley and Sons, New York1993: 100-120Google Scholar). An overnight culture of BL21(DE3)/hDHFR F31R/Q35E-pET24 was used to inoculate 1 liter of LB medium. The culture was grown at 37 °C until the A600 nm reached ≈0.7. Protein expression was induced with the addition of 1 mm of isopropyl 1-thio-β-d-galactopyranoside, after which the cells were grown for 16 h at 22 °C. Induced cells were harvested by centrifugation (4000 × g for 30 min at 4 °C). The cell pellet was resuspended in 10 mm Tris-HCl, pH 8.3, at 4 °C. The cells were lysed on ice using a Branson sonicator (four pulses at 200 watts for 30 s with a tapered micro-tip). The cellular debris was pelleted by centrifugation (4000 × g for 30 min at 4 °C), and the supernatant was filtered through a 0.2-μm filter before purification. Purification was performed following a two-step purification protocol on an AKTA fast-protein liquid chromatography (Amersham Biosciences) at 5 °C. First, the supernatant was applied to an anion-exchange DEAE-Sepharose column (1.6 × 30 cm) followed by a 3-column volume wash with 10 mm Tris-HCl, pH 8.3, at 2 ml/min. A linear gradient of 5 column volumes with NaCl (0–200 mm) in 10 mm Tris-HCl, pH 8.3, was used to elute the F31R/Q35E variant. hDHFR activity was monitored in MATS buffer, pH 7.6, in the presence of 100 μm each of NADPH and DHF. Activity was measured in flat-bottom plates (Costar #3595) by monitoring concurrent depletion of NADPH and DHF (Δϵ340 nm = 12 800 m−1 cm−1) on a FLUOstar OPTIMA UV-visible plate reader (BMG Laboratories, Offenburg, Germany). Active fractions were pooled and dialyzed overnight at 4 °C against 50 mm phosphate buffer, pH 7.5. Following dialysis, the sample (45 ml) was concentrated to 1.5 ml using an Amicon concentrator (molecular weight cut-off 10000, Millipore), for injection on a Superose 12 column (1.6 × 55 cm). The sample was eluted with 50 mm phosphate buffer, pH 7.5, at a flow rate of 1.5 ml/min. hDHFR activity was monitored as described above. Enzyme purity was evaluated using separation by SDS-PAGE (15% (w/v) polyacrylamide gel) stained by the zinc-imidazole method (31Fernandez-Patron C. Castellanos-Serra L. Rodriguez P. BioTechniques. 1992; 12: 564-573PubMed Google Scholar) and quantified using the public domain image analysis software Scion Image (NIH, rsb.info.nih.gov/nih-image). Protein concentration was quantified using the Bradford assay (Bio-Rad). Purified hDHFR F31R/Q35E enzyme was concentrated to 10 mg/ml using an Amicon concentrator (molecular weight cut-off 10000). MTX and NADPH were prepared as described previously (18Volpato J.P. Fossati E. Pelletier J.N. J. Mol. Biol. 2007; 373: 599-611Crossref PubMed Scopus (31) Google Scholar) and were added at a final concentration of 2 mm each (5-fold molar excess) to the protein sample. Crystallization experiments were set up using hanging drop, vapor-diffusion experiments, with a reservoir volume of 1 ml and a drop size of 4 μl of equal volumes of protein and reservoir solutions. A reservoir solution containing 0.2 m cadmium phosphate and 2.2 m ammonium sulfate yielded crystal-like formations that diffracted poorly. These crystals were crushed using a Hampton Seed Bead Kit and used as seeds (1/10 dilution). Rod-shaped crystals were obtained from crystallization experiments with 0.2 m sodium phosphate and 2.2 m ammonium sulfate as the reservoir solution and with drops containing 1.5 μl of protein, 2 μl of reservoir solution, and 0.5 μl of seeding solution. The crystals were soaked in the mother liquor supplemented with 15% glycerol as a cryoprotectant and frozen in a nitrogen cryostream (model X-stream 2000). Data were collected using a Rigaku RU-H3R generator, equipped with Osmic focusing mirrors, and an R-axis IV++ image plate detector, and processed using HKL-2000 (32Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 286-306Crossref PubMed Scopus (18) Google Scholar) (Table 1).TABLE 1Crystallographic dataData collection statistics Space groupP212121 Number of molecules per asymmetric unit1a (Å)42.348b (Å)47.868c (Å)90.715α = β = γ (°)90 Wavelength (Å)1.5418 Resolution range (Å)aItems in parentheses refer to the highest resolution shell.1.70–10.52 (1.70–1.76) Completeness (%)aItems in parentheses refer to the highest resolution shell.97.2 (93.9) RedundancyaItems in parentheses refer to the highest resolution shell.11.7 (9.5) Rmerge (%)aItems in parentheses refer to the highest resolution shell.6.4 (47.3)Refinement statistics Total number of reflections (reflections in R-free set)20,304 (2,066) Rfactor (%)17.93 Rfree (10% free test set) (%)22.30 Number of atoms1719Protein1507Water138Ions41Inhibitor33 r.m.s.d.Bond length (Å)0.012Bond angle (°)1.417 Average atomic B-factor (Å2)16.372Protein (Å2)14.99Water (Å2)30.57Ions (Å2)35.20Inhibitor (Å2)15.78 Wilson B-factor (Å2)21.585 Luzzati sigma A coordinate error (observed) (Å)0.12 Luzzati sigma A coordinate error (R-free set) (Å)0.11 Ramachandran plot (non-Gly, non-Pro residues)159 (100%)Residues in favored positions146 (91.8%)Residues in allowed positions13 (8.2%)Residues in disallowed positions0 (0%)a Items in parentheses refer to the highest resolution shell. Open table in a new tab The structure was determined by molecular replacement using Phaser (33Storoni L.C. McCoy A.J. Read R.J. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 432-438Crossref PubMed Scopus (1092) Google Scholar), which found a single protein molecule in the asymmetric unit (Resolution Range Used: 1.70–25.99; Log Likelihood Gain (refined): 726.123). A lower quality His-tagged F31R/Q35E-MTX-NADPH structure (see supplemental materials) was used as a molecular replacement model. Reciprocal-space refinement was performed using Refmac (34Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13811) Google Scholar) and included individual isotropic B-factor refinement as well as TLS refinement in the final stages of refinement. Manual model building was performed periodically using Coot (35Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22994) Google Scholar) (Table 1). The ligands were prepared as pdb files using ChemDraw 8.0 and Chem3D 8.0 (CambridgeSoft, Cambridge, MA). Energy minimization of ligands was performed with the integrated MM2 energy minimization script in Chem3D. Automated docking experiments were performed using the Autodock 4 software package (Scripps Research Institute, La Jolla, CA). Macromolecules PDB ID 1U72 and 3EIG were stripped of all ligands and heteroatoms, with the exception of the highly conserved active site water molecule (H2O #216 in 1U72, H2O #244 in 3EIG), and were prepared using default settings. A box covering the entire folate binding site and more than half the NADPH binding site was generated as a docking grid. 50 runs of a Lamarckian genetic algorithm using default settings were performed. Following docking, clusters were evaluated according to total binding energies calculated by Autodock 4, and the minimal energy conformation within the lowest energy cluster was retained for comparison with crystal structures. To determine the effect of each constituent amino acid substitution of the F31R/Q35E variant on DHF and MTX binding, the singly-substituted F31R and Q35E were created and their kinetic and inhibition parameters were determined (Table 2). The reactivity (kcat) of F31R and Q35E are 5-fold lower (1.9 ± 0.3 s−1) and 2-fold lower (4.6 ± 0.2 s−1) than WT hDHFR, respectively. The kcat of F31R/Q35E is similar to that of the F31R variant, indicating that loss of reactivity in F31R/Q35E is primarily due to the F31R substitution. The Michaelis constants (KmDHF) of variants F31R and Q35E are 110 ± 60 and 250 ± 90 nm, respectively, illustrating a slight decrease in DHF affinity (1.5- and 3-fold, respectively) relative to the WT. The high % error on KmDHF results from the low values of KmDHF and the correspondingly low spectrophotometric signal, which was enhanced by the use of 10-cm path length cuvettes. Kinetic data for the F31R variant had previously been reported (10Patel M. Sleep S.E. Lewis W.S. Spencer H.T. Mareya S.M. Sorrentino B.P. Blakley R.L. Hum. Gene Ther. 1997; 8: 2069-2077Crossref PubMed Scopus (27) Google Scholar), and compares well with our data, although we determined a KmDHF value that is 6-fold lower than previously reported. Because we expected a low value for KmDHF and a correspondingly low spectrophotometric signal, we used 10-cm cuvettes in KmDHF determination, rather than 1-cm cuvettes (10Patel M. Sleep S.E. Lewis W.S. Spencer H.T. Mareya S.M. Sorrentino B.P. Blakley R.L. Hum. Gene Ther. 1997; 8: 2069-2077Crossref PubMed Scopus (27) Google Scholar). This enabled a more precise measurement in the target range. Inhibition constants for MTX (KiMTX) revealed that the F31R substitution (KiMTX = 1.1 nm) confers a 35-fold loss in MTX affinity, whereas the Q35E substitution (KiMTX = 0.048 nm) displays a modest decrease of MTX affinity relative to WT (1.5-fold decrease). Like the F31R variant, the F31R/Q35E variant displayed larger decreases in MTX affinity than DHF affinity relative to the WT. However, the Q35E substitution modestly decreased DHF affinity, while having a negligible effect on MTX affinity. The F31R/Q35E variant presented both of these features, as DHF affinity decreased 9-fold relative to WT, whereas MTX affinity decreased >650-fold relative to the WT. Thus, addition of the Q35E substitution to variant F31R increased the KmDHF of F31R 6-fold while increasing the KiMTX of F31R nearly 20-fold.TABLE 2Kinetic and inhibitory parameters of WT hDHFR and hDHFR variant F31R/Q35EVariantkcatKmDHFkcat/KmDHFΔΔGDHFaΔΔG = −RT × ln(KmDHF WT/KmDHF variant); T = 293 K.IC50MTXKiMTXΔΔGMTXbΔΔG = −RT × ln(KiMTX WT/KiMTX variant); T = 293 K.s−1nms−1 μm−1kcal/molnmnmkcal/molWTcValues were taken from Refs. (16, 18).10 ± 2 130041 ± 14<0.0310F31R1.9 ± 0.3110 ± 6017 ± 120.31100 ± 6001.1 ± 0.62.1Q35E4.6 ± 0.2250 ± 9018 ± 70.719 ± 40.048 ± 0.0090.3F31R/Q35EcValues were taken from Refs
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