A nuclear magnetic resonance study of nicotinamide adenine dinucleotide phosphate binding to Lactobacillus casei dihydrofolate reductase.

The binding of NADP+ to dihydrofolate reductase (EC 1.5.1.3) in the presence and absence of substrate analogs has been studied using 1H and 13C nuclear magnetic resonance (NMR). NADP+ binds strongly to the enzyme alone and in the presence of folate, aminopterin, and methotrexate with a stoichiometry of 1 mol of NADP+/mol of enzyme. In the 13C spectra of the binary and ternary complexes, separate signals were observed for the carboxamide carbon of free and bound [13CO]NADP+ (enriched 90% in 13C). The 13C signal of the NADP+-reductase complex is much broader than that in the ternary complex with methotrexate because of exchange line broadening on the binary complex signal. From the difference in line widths (17.5 +/- 3.0 Hz) an estimate of the dissociation rate constant of the binary complex has been obtained (55 +/- 10 sec-1). The dissociation rate of the NADP+-reductase complex is not the rate-limiting step in the overall reaction. In the various complexes studied large 13C chemical shifts were measured for bound [13CO]NADP+ relative to free NADP+ (upfield shifts of 1.6-4.3 ppm). The most likely origin of the bound shifts lies in the effects on the shieldings of electric fields from nearby charged groups. For the NADP+-reductase-folate system two 13C signals from bound NADP+ are observed indicating the presence of more than one form of the ternary complex. The IH spectra of the binary and ternary complexes confirm both the stoichiometry and the value of the dissociation rate constant obtained from the 13C experiments. Substantial changes in the IH spectrum of the protein were observed in the different complexes and these are distinct from those seen in the presence of NADPH.     

Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+

Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.     

Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle

Escherichia coli dihydrofolate reductase (DHFR) has several flexible loops surrounding the active site that play a functional role in substrate and cofactor binding and in catalysis. We have used heteronuclear NMR methods to probe the loop conformations in solution in complexes of DHFR formed during the catalytic cycle. To facilitate the NMR analysis, the enzyme was labeled selectively with [(15)N]alanine. The 13 alanine resonances provide a fingerprint of the protein structure and report on the active site loop conformations and binding of substrate, product, and cofactor. Spectra were recorded for binary and ternary complexes of wild-type DHFR bound to the substrate dihydrofolate (DHF), the product tetrahydrofolate (THF), the pseudosubstrate folate, reduced and oxidized NADPH cofactor, and the inactive cofactor analogue 5,6-dihydroNADPH. The data show that DHFR exists in solution in two dominant conformational states, with the active site loops adopting conformations that closely approximate the occluded or closed conformations identified in earlier X-ray crystallographic analyses. A minor population of a third conformer of unknown structure was observed for the apoenzyme and for the disordered binary complex with 5,6-dihydroNADPH. The reactive Michaelis complex, with both DHF and NADPH bound to the enzyme, could not be studied directly but was modeled by the ternary folate:NADP(+) and dihydrofolate:NADP(+) complexes. From the NMR data, we are able to characterize the active site loop conformation and the occupancy of the substrate and cofactor binding sites in all intermediates formed in the extended catalytic cycle. In the dominant kinetic pathway under steady-state conditions, only the holoenzyme (the binary NADPH complex) and the Michaelis complex adopt the closed loop conformation, and all product complexes are occluded. The catalytic cycle thus involves obligatory conformational transitions between the closed and occluded states. Parallel studies on the catalytically impaired G121V mutant DHFR show that formation of the closed state, in which the nicotinamide ring of the cofactor is inserted into the active site, is energetically disfavored. The G121V mutation, at a position distant from the active site, interferes with coupled loop movements and appears to impair catalysis by destabilizing the closed Michaelis complex and introducing an extra step into the kinetic pathway.     

Crystal structure of chicken liver dihydrofolate reductase complexed with NADP+ and biopterin.

The 2.2-A crystal structure of chicken liver dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a ternary complex with NADP+ and biopterin (a poor substrate). The space group and unit cell are isomorphous with the previously reported structure of chicken liver DHFR complexed with NADPH and phenyltriazine [Volz, K. W., Matthews, D. A., Alden, R. A., Freer, S. T., Hansch, C., Kaufman, B. T., & Kraut, J. (1982) J. Biol. Chem. 257, 2528-2536]. The structure contains an ordered water molecule hydrogen-bonded to both hydroxyls of the biopterin dihydroxypropyl group as well as to O4 and N5 of the biopterin pteridine ring. This water molecule, not observed in previously determined DHFR structures, is positioned to complete a proposed route for proton transfer from the side-chain carboxylate of E30 to N5 of the pteridine ring. Protonation of N5 is believed to occur during the reduction of dihydropteridine substrates. The positions of the NADP+ nicotinamide and biopterin pteridine rings are quite similar to the nicotinamide and pteridine ring positions in the Escherichia coli DHFR.NADP+.folate complex [Bystroff, C., Oatley, S. J., & Kraut, J. (1990) Biochemistry 29, 3263-3277], suggesting that the reduction of biopterin and the reduction of folate occur via similar mechanisms, that the binding geometry of the nicotinamide and pteridine rings is conserved between DHFR species, and that the p-aminobenzoylglutamate moiety of folate is not required for correct positioning of the pteridine ring in ground-state ternary complexes. Instead, binding of the p-aminobenzoylglutamate moiety of folate may induce the side chain of residue 31 (tyrosine or phenylalanine) in vertebrate DHFRs to adopt a conformation in which the opening to the pteridine binding site is too narrow to allow the substrate to diffuse away rapidly. A reverse conformational change of residue 31 is proposed to be required for tetrahydrofolate release.     

NMR studies of the interaction of a type II dihydrofolate reductase with pyridine nucleotides reveal unexpected phosphatase and reductase activity

The interaction of type II R67 dihydrofolate reductase (DHFR) with its cofactor nicotinamide adenine dinucleotide phosphate (NADP(+)) has been studied using nuclear magnetic resonance (NMR). Doubly labeled [U-(13)C,(15)N]DHFR was obtained from Escherichia coli grown on a medium containing [U-(13)C]-D-glucose and (15)NH(4)Cl, and the 16 disordered N-terminal amino acids were removed by treatment with chymotrypsin. Backbone and side chain NMR assignments were made using triple-resonance experiments. The degeneracy of the amide (1)H and (15)N shifts of the tetrameric DHFR was preserved upon addition of NADP(+), consistent with kinetic averaging among equivalent binding sites. Analysis of the more titration-sensitive DHFR amide resonances as a function of added NADP(+) gave a K(D) of 131 +/- 50 microM, consistent with previous determinations using other methodology. We have found that the (1)H spectrum of NADP(+) in the presence of the R67 DHFR changes as a function of time. Comparison with standard samples and mass spectrometric analysis indicates a slow conversion of NADP(+) to NAD(+), i.e., an apparent NADP(+) phosphatase activity. Studies of this activity in the presence of folate and a folate analogue support the conclusion that this activity results from an interaction with the DHFR rather than a contaminating phosphatase. (1)H NMR studies of a mixture of NADP(+) and NADPH in the presence of the enzyme reveal that a ternary complex forms in which the N-4A and N-4B nuclei of the NADPH are in the proximity of the N-4 and N-5 nuclei of NADP(+). Studies using the NADP(+) analogue acetylpyridine adenosine dinucleotide phosphate (APADP(+)) demonstrated a low level of enzyme-catalyzed hydride transfer from NADPH. Analysis of DHFR backbone dynamics revealed little change upon binding of NADP(+). These additional catalytic activities and dynamic behavior are in marked contrast to those of type I DHFR.     

Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes

Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution. (1)H(N), (15)N, and (13)C(alpha) resonance assignments have been made for the ternary complex with folate and oxidized NADP(+) cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6-dihydroNADPH. The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme). Analysis of (1)H(N) and (15)N chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme. Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide-ribose binding sites of the NADPH cofactor. The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X-ray crystallography; there is no evidence that the open conformation observed in some X-ray structures of E. coli DHFR are populated in solution.     

Interligand Overhauser effects in type II dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria as a resistance mechanism to the increased clinical use of the antibacterial drug trimethoprim. Type II DHFRs are not homologous in either sequence or structure with chromosomal DHFRs. The type II enzymes contain four identical subunits which form a homotetramer containing a single active site pore accessible from either end. Although the crystal structure of the complex of R67 DHFR with folate has been reported [Narayana et al. (1995) Nat. Struct. Biol. 2, 1018], the nature of the ternary complex which must form with substrate and cofactor is unclear. We have performed transferred NOE and interligand NOE (ILOE) studies to analyze the ternary complexes formed from NADP(+) and folate in order to probe the structure of the ternary complex. Consistent with previous studies of the binary complex formed from another type II DHFR, the ribonicotinamide bond of NADP(+) was found to adopt a syn conformation, while the adenosine moiety adopts an anti conformation. Large ILOE peaks connecting NADP(+) H4 and H5 with folate H9 protons are observed, while the absence of a large ILOE connecting NADP(+) H4 and H5 with folate H7 indicates that the relative orientation of the two ligands differs significantly from the orientation in the chromosomal enzyme. To obtain more detailed insight, we prepared and studied the folate analogue 2-deamino-2-methyl-5,8-dideazafolate (DMDDF) which contains additional protons in order to provide additional NOEs. For this analogue, the exchange characteristics of the corresponding ternary complex were considerably poorer, and it was necessary to utilize higher enzyme concentrations and higher temperature in order to obtain ILOE information. The results support a structure in which the NADP(+) and folate/DMDDF molecules extend in opposite directions parallel to the long axis of the pore, with the nicotinamide and pterin ring systems approximately stacked at the center. Such a structure leads to a ternary complex which is in many respects similar to the gas-phase theoretical calculations of the dihydrofolate-NADPH transition state by Andres et al. [(1996) Bioorg. Chem. 24, 10-18]. Analogous NMR studies performed on folate, DMDDF, and R67 DHFR indicate formation of a ternary complex in which two symmetry-related binding sites are occupied by folate and DMDDF.     

Methotrexate binds in a non-productive orientation to human dihydrofolate reductase in solution, based on NMR spectroscopy

Dihydrofolate reductase (DHFR) is an intracellular target enzyme for folate antagonist drugs, including methotrexate. In order to compare the binding of methotrexate to human DHFR in solution with that observed in the crystalline state, NMR spectroscopy has been used to determine the conformation of the drug bound to human DHFR in solution. In agreement with what has been observed in the crystalline state, NOE's identified protein and methotrexate protons indicate that methotrexate binds in a non-productive orientation. In contrast to what has been reported for E. coli DHFR in solution, only one bound conformation of methotrexate is observed.     

15N and 1H NMR evidence for multiple conformations of the complex of dihydrofolate reductase with its substrate, folate

The binding of folate to Lactobacillus casei dihydrofolate reductase in the presence and absence of NADP+ has been studied by 15N NMR, using [5-15N]folate. In the presence of NADP+, three separate signals were observed for the single 15N atom, in agreement with our earlier evidence from 1H and 13C NMR for multiple conformations of this complex [(1982) Biochemistry 21, 5831-5838]. The 15N spectra of the binary enzyme-folate complex provide evidence for the first time that this complex also exists in at least two conformational states. This is confirmed by the observation of two separate resonances for the 7-proton of bound folate, located by two-dimensional exchange spectroscopy.     

Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution

The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been determined at atomic resolution. The potent inhibitors 6-([5-quinolylamino]methyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9439) and (Z)-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9662) were developed at Southern Research Institute against Toxoplasma gondii DHFR-thymidylate synthase. The 5-deazapteridine ring of each inhibitor adopts an unusual puckered conformation that enables the formation of identical contacts in the active site. Conversely, the quinoline and dimethoxybenzene moieties exhibit distinct binding characteristics that account for the differences in inhibitory activity. In both structures, a salt-bridge is formed between Arg70 in the active site and Glu44 from a symmetry-related molecule in the crystal lattice that mimics the binding of methotrexate to DHFR.     

Effect of cofactor binding and loop conformation on side chain methyl dynamics in dihydrofolate reductase

Dihydrofolate reductase (DHFR) has several flexible active site loops that facilitate ligand binding and catalysis. Previous studies of backbone dynamics in several complexes of DHFR indicate that the time scale and amplitude of motion depend on the conformation of the active site loops. In this study, information on dynamics is extended to methyl-containing side chains. To understand the role of side chain dynamics in ligand binding and loop conformation, methyl deuterium relaxation rates of Escherichia coli DHFR in binary folate and ternary folate:NADP+ complexes have been measured, together with chi(1) rotamer populations for threonine, isoleucine, and valine residues, determined from measurements of 3J(CgammaCO) and 3J(CgammaN) coupling constants. The results indicate that, in addition to backbone motional restriction in the adenosine-binding site, side chain flexibility in the active site and the surrounding active site loops is diminished upon binding NADP+. Resonances for several methyls in the active site and the surrounding active site loops were severely broadened in the folate:NADP+ ternary complex, suggesting the presence of motion on the chemical shift time scale. The side chains of Ile14 and Ile94, which pack against the nicotinamide and pterin rings of the cofactor and substrate, respectively, exhibit rotamer disorder in the ternary folate:NADP+ complex. Conformational fluctuations of these side chains may play a role in transition state stabilization; the observed line broadening for Ile14 suggests motions on a microsecond/millisecond time scale.     

NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21----Leu mutant indicate that the delicately poised equilibria can be perturbed. For example, in the case of the ternary complex formed between enzyme, trimethoprim, and NADP+, two almost equally populated conformations (forms I and II) are seen with the wild-type enzyme but only form II (the one in which the nicotinamide ring of the coenzyme is extended away from the enzyme structure and into the solvent) is observed for the mutant enzyme complex. It appears that the Trp 21----Leu substitution has a major effect on the binding of the nicotinamide ring of the coenzyme. For the Asp 26----Glu enzyme there is a change in the bound conformation of the substrate folate. Further indications that some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim come from the observation of a change in the dynamics of the bound trimethoprim molecule as seen from the increased rate of the flipping of the 13C-labeled benzyl ring and the increased rate of the N1-H bond breaking.     

Nuclear magnetic resonance study of the state of protonation of inhibitors bound to mutant dihydrofolate reductase lacking the active-site carboxyl

13C nuclear magnetic resonance spectra have been obtained for complexes of [2-13C]methotrexate and [2-13C]trimethoprim with wild-type dihydrofolate reductase (DHFR) from Escherichia coli and with two mutant enzymes in which aspartic acid-27 is replaced by asparagine and by serine, respectively. In both the wild-type and mutated enzymes, exchange between the free inhibitor and the enzyme-complexed inhibitor is slow on the NMR time scale; hence, despite the considerably increased dissociation constants for binary complexes with the enzymes, the dissociation rate remains small relative to the frequency separation of the resonances. In all cases but one, the pKa of an inhibitor that is complexed to enzyme differs greatly from that of the free inhibitor. However, while the pKa of both inhibitors in complexes with the wild-type enzyme is elevated to above 10, the pKa of the inhibitors complexed with the Asn-27 and Ser-27 enzymes is lowered to a value below 4. Exact determinations of bound pKa values are limited by the solubility of the enzyme and the dissociation constants of the complexes. The single exception to these general conclusions is the ternary complex of the Ser-27 DHFR with trimethoprim and NADPH. In this complex, both free and enzyme-complexed trimethoprim exhibit similar pKa values (approximately equal to 7.6). However, both the exchange between free and enzyme-complexed inhibitor and the protonation of the enzyme-complexed inhibitor are slow in the NMR time scale, so that the spectra reveal three resonances corresponding to free inhibitor, to protonated enzyme-complexed inhibitor, and to unprotonated enzyme-complexed inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circular dichroism studies of dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428: ternary complexes.

Circular dichroism has been used to monitor the binding of pyridine nucleotide cofactors to enzyme-folate analog complexes of dihydrofolate reductase from Escherichia coli B (MB 1428). The enzyme binds one molar equivalent of many folate analogs and two molar equivalents of several pyridine nucleotide cofactors. The apo-enzyme has very low optical activity. The binding of folate analogs including folate, dihydrofolate, methotrexate, trimethoprim and pyrimethamine induce large Cotton effects. Pyridine nucleotides when bound to the enzyme-folate analog complexes also induce new optically active bands; all the effects being due to the first molar equivalent of cofactor bound. NADPH and NADP+ induce very similar bands when bound to the enzyme-methotrexate complex suggesting that the geometry of the complexes formed are very similar. The oxidized and reduced cofactor likewise have similar effects on the enzyme-folate complex. However, NADPH and NADP+ addition to both the enzyme-trimethoprim and enzyme-pyrimethamine complexes have significantly different effects on the circular dichroism spectra, suggesting that the inhibitors which are less homologous to the natural dihydrofolate substrate allow more conformational freedom in the enzyme-inhibitor-cofactor complex. In most cases the prior binding of the folate analog greatly increases the binding of the first molar equivalent of cofactor so that at concentrations of approx. 5-20 muM the binding appears stoichiometric. Pyrimethamine is an exception in that it apparently has no effect on the binding of NADPH to the enzyme.     

Characterization of rates of ring-flipping in trimethoprim in its ternary complexes with Lactobacillus casei dihydrofolate reductase and coenzyme analogues

NMR measurements have been used to investigate rates of ring-flipping and the activation parameters for the trimethoxybenzyl ring of the antibacterial drug trimethoprim (TMP) bound to Lactobacillus casei dihydrofolate reductase (DHFR) for a series of ternary complexes formed with analogues of the coenzyme NADPH. Rates were obtained at several temperatures from line shape analyses ((13)C-edited HSQC (1)H spectra) and transfer of magnetization measurements (zz-HSQC) on complexes containing 3'-O-[(13)C]trimethoprim. Examination of the structures of the complexes indicates that ring-flipping can only be achieved following major conformational changes and transient fluctuations of the protein and coenzyme structure around the trimethoxybenzyl ring. There is no simple correlation between rates of ring-flipping and binding constants. The presence of the coenzyme nicotinamide ring (in either its reduced or its oxidized forms) in the binding site close to the trimethoxybenzyl ring moiety is the major factor reducing the ring-flipping on coenzyme binding. Thus, the ternary complex with NADPH shows the largest reduction in the rate of ring-flipping (11 +/- 3 s(-)(1) at 298 K) as compared with the binary complex (793 +/- 80 s(-)(1) at 298 K). Complexes with NADPH analogues that either have no nicotinamide ring or are known to have their nicotinamide rings removed from the binding site show the smallest reductions. For the DHFR.TMP.NADP(+) complex where there are two conformations present, very different rates of ring-flipping were observed for the two forms. The activation parameters (DeltaH() and DeltaS()) for the ring-flipping in all the complexes are discussed in terms of the protein-ligand interactions and the possible constraints on the pathway through the transition state.     

Three-dimensional structure of M. tuberculosis dihydrofolate reductase reveals opportunities for the design of novel tuberculosis drugs

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate and is essential for the synthesis of thymidylate, purines and several amino acids. Inhibition of the enzyme's activity leads to arrest of DNA synthesis and cell death. The enzyme has been studied extensively as a drug target for bacterial, protozoal and fungal infections, and also for neoplastic and autoimmune diseases. Here, we report the crystal structure of dihydrofolate reductase from Mycobacterium tuberculosis, a human pathogen responsible for the death of millions of human beings per year. Three crystal structures of ternary complexes of M. tuberculosis DHFR with NADP and different inhibitors have been determined, as well as the binary complex with NADP, with resolutions ranging from 1.7 to 2.0 A. The three DHFR inhibitors are the anticancer drug methotrexate, the antimicrobial trimethoprim and Br-WR99210, an analogue of the antimalarial agent WR99210. Structural comparison of these complexes with human dihydrofolate reductase indicates that the overall protein folds are similar, despite only 26 % sequence identity, but that the environments of both NADP and of the inhibitors contain interesting differences between the enzymes from host and pathogen. Specifically, residues Ala101 and Leu102 near the N6 of NADP are distinctly more hydrophobic in the M. tuberculosis than in the human enzyme. Another striking difference occurs in a region near atoms N1 and N8 of methotrexate, which is also near atom N1 of trimethoprim, and near the N1 and two methyl groups of Br-WR99210. A glycerol molecule binds here in a pocket of the M. tuberculosis DHFR:MTX complex, while this pocket is essentially filled with hydrophobic side-chains in the human enzyme. These differences between the enzymes from pathogen and host provide opportunities for designing new selective inhibitors of M. tuberculosis DHFR.     

Crystal structures of Escherichia coli dihydrofolate reductase: the NADP+ holoenzyme and the folate.NADP+ ternary complex. Substrate binding and a model for the transition state

The crystal structure of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli has been solved as the binary complex with NADP+ (the holoenzyme) and as the ternary complex with NADP+ and folate. The Bragg law resolutions of the structures are 2.4 and 2.5 A, respectively. The new crystal forms are nonisomorphous with each other and with the methotrexate binary complex reported earlier [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. In general, NADP+ and folate binding conform to predictions, but the nicotinamide moiety of NADP+ is disordered in the holoenzyme and ordered in the ternary complex. A mobile loop (residues 16-20) involved in binding the nicotinamide is also disordered in the holoenzyme. We report a detailed analysis of the binding interactions for both ligands, paying special attention to several apparently strained interactions that may favor the transition state for hydride transfer. Hypothetical models are presented for the binding of 7,8-dihydrofolate in the Michaelis complex and for the transition-state complex.     

Multinuclear NMR characterization of two coexisting conformational states of the Lactobacillus casei dihydrofolate reductase-trimethoprim-NADP+ complex

The complex of Lactobacillus casei dihydrofolate reductase with trimethoprim and NADP+ exists in solution as a mixture of approximately equal amounts of two slowly interconverting conformational states [Gronenborn, A., Birdsall, B., Hyde, E. I., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1981) Mol. Pharmacol. 20, 145]. These have now been further characterized by multinuclear NMR experiments, and a partial structural model has been proposed. 1H NMR spectra at 500 MHz show that the environments of six of the seven histidine residues differ between the two conformations. The characteristic 1H and 31P chemical shifts of nuclei of the coenzyme in the two conformations of the complex are identical in analogous complexes formed with a number of trimethoprim analogues, indicating that the nature of the two conformations is the same in each case. The pyrophosphate 31P resonances have been assigned to the two conformations, and integration of the 31P spectrum shows that the ratio of conformation I to conformation II varies from 0.4 to 2.3 in the complexes with the various trimethoprim analogues, the ratio for the trimethoprim complex itself being 1.2. Transferred NOE experiments, together with the 1H and 13C chemical shifts, indicate that in conformation II of the complex the nicotinamide ring of the coenzyme has swung away from the enzyme surface into solution; this is made possible by changes in the conformation of the pyrophosphate moiety. In conformation I, by contrast, the nicotinamide ring remains bound to the enzyme. 13C and 15N experiments show that trimethoprim is protonated on N1 in both conformations of the ternary complex. Analysis of the 1H chemical shifts of trimethoprim in terms of ring current effects shows that in conformation I of the ternary complex trimethoprim retains the same conformation as in its binary complex, but 13C, 15N, and 19F [using 2,4-diamino-5-(3,5-dimethoxy-4-fluoro-benzyl)pyrimidine] experiments show that the environment of both the pyrimidine ring and benzyl ring is affected by the proximity of the coenzyme. Less information is available about the conformation of the inhibitor in conformation II of the complex, but its environment is similar to that in the binary enzyme-inhibitor complex. The implications of the existence of these two conformations of the enzyme for understanding cooperativity in binding between NADP+ and trimethoprim are briefly discussed.     

Role of water in the catalytic cycle of E. coli dihydrofolate reductase

Dihydrofolate reductase (DHFR) catalyzes the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of 7,8-dihydrofolate (H2F) to 5,6,7,8-tetrahydrofolate (H4F). Because of the absence of any ionizable group in the vicinity of N5 of dihydrofolate it has been proposed that N5 could be protonated directly by a water molecule at the active site in the ternary complex of the Escherichia coli enzyme with cofactor and substrate. However, in the X-ray structures representing the Michaelis complex of the E. coli enzyme, a water molecule has never been observed in a position that could allow protonation of N5. In fact, the side chain of Met 20 blocks access to N5. Energy minimization reported here revealed that water could be placed in hydrogen bonding distance of N5 with only minor conformational changes. The r.m.s. deviation between the conformation of the M20 loop observed in the crystal structures of the ternary complexes and the conformation adopted after energy minimization was only 0.79 A. We performed molecular dynamics simulations to determine the accessibility by water of the active site of the Michaelis complex of DHFR. Water could access N5 relatively freely after an equilibration time of approximately 300 psec during which the side chain of Met 20 blocked water access. Protonation of N5 did not increase the accessibility by water. Surprisingly the number of near-attack conformations, in which the distance between the pro-R hydrogen of NADPH and C6 of dihydrofolate was less than 3.5 A and the angle between C4 and the pro-R hydrogen of NADPH and C6 of dihydrofolate was greater than 120 degrees, did not increase after protonation. However, when the hydride was transferred from NADPH to C6 of dihydrofolate before protonation, the side chain of Met 20 moved away from N5 after approximately 100 psec thereby providing water access. The average time during which water was found in hydrogen bonding distance to N5 was significantly increased. These results suggest that hydride transfer might occur early to midway through the reaction followed by protonation. Such a mechanism is supported by the very close contact between C4 of NADP+ and C6 of folate observed in the crystal structures of the ternary enzyme complexes, when the M20 loop is in its closed conformation.