Dihydrofolate reductase. The stereochemistry of inhibitor selectivity

X-ray structural results are reported for 10 triazine and pyrimidine inhibitors of dihydrofolate reductase, each one studied as a ternary complex with NADPH and chicken dihydrofolate reductase. Analysis of these data and comparison with structural results from the preceding paper (Matthews, D.A., Bolin, J.T., Burridge, J.M., Filman, D.J., Volz, K.W., Kaufman, B. T., Beddell, C.R., Champness, J.N., Stammers, D.K., and Kraut, J. (1985) J. Biol. Chem. 260, 381-391) in which we contrasted binding of the antibiotic trimethoprim (TMP) to chicken dihydrofolate reductase on the one hand with its binding to Escherichia coli dihydrofolate reductase on the other, permit identification of differences that are important in accounting for TMP's selectivity. The crystallographic evidence strongly suggests that loss of a potential hydrogen bond between the 4-amino group of TMP and the backbone carbonyl of Val-115 when TMP binds to chicken dihydrofolate reductase but not when it binds to the E. coli reductase is the major factor responsible for this drug's more potent inhibition of bacterial dihydrofolate reductase. A key finding of the current study which is important in understanding why TMP binds differently to chicken and E. coli dihydrofolate reductases is that residues on opposite sides of the active-site cleft in chicken dihydrofolate reductase are about 1.5-2.0 A further apart than are structurally equivalent residues in the E. coli enzyme.     

Influence of environment on the antifolate drug trimethoprim: energy minimization studies

Environmental effects on trimethoprim (TMP), an inhibitor of bacterial dihydrofolate reductase (DHFR), were investigated with energy minimizations in vacuo, in the crystal, and in aqueous solution. The conformations, harmonic dynamics, and energetics of the antibacterial drug calculated in these environments were compared with each other and with those of two enzyme-bound drugs. Valence and torsion angles and their energies and overall intra- and intermolecular energies compensated one another in the minimized TMP structures. The conformations of the isolated and aqueous molecules were similar to that of TMP bound to chicken liver DHFR, while the structures from the TMP crystal and from the Escherichia coli DHFR complex were unique. Since neither the small-molecule crystal nor a local minimum of the isolated molecule gave the conformation of TMP bound to the bacterial enzyme, a combination of several experimental and theoretical techniques may be necessary to probe accessible conformations of a molecule.     

Nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase.

The resonances of the aromatic protons of trimethoprim [2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine] in its complexes with dihydrofolate reductases from Lactobacillus casei and Escherichia coli cannot be directly observed. Their chemical shifts have been determined by transfer of saturation experiments and by difference spectroscopy using [2',6'-2H2]trimethoprim. The complex of 2,4-diamino-5-(3',4'-dimethoxy-5'-bromobenzyl)pyrimidine with the L. casei enzyme has also been examined. At room temperature, the 2',6'-proton resonance of bound trimethoprim is very broad (line width great than 30 Hz); with the E. coli enzyme, the resonance sharpens with increasing temperature so as to be clearly visible by difference spectroscopy at 45 degrees C. This line broadening is attributed to an exchange contribution, arising from the slow rate of "flipping" about the C7-C1' bond of bound trimethoprim. The transfer of saturation measurements were also used to determine the dissociation rate constants of the complexes. In the course of these experiments, a decrease in intensity of the resonance of the 2',6'-proton resonance of free trimethoprim on irradiation at the resonance of the 6 proton of free trimethoprim was observed, which only occurred in the presence of the enzyme. This is interpreted as a nuclear Overhauser effect between two protons of the bound ligand transferred to those of the free ligand by the exchange of the ligand between the two states. The chemical shift changes observed on the binding of trimethoprim to dihydrofolate reductase are interpreted in terms of the ring-current shift contributions from the two aromatic rings of trimethoprim and from that of phenylalanine-30. On the basis of this analysis of the chemical shifts, a model for the structure of the enzyme-trimethoprim complex is proposed. This model is consistent with the (indirect) observation of a nuclear Overhauser effect between the 2',6' and 6 protons of bound trimethoprim.     

Species-specific irreversible inhibition of Neisseria gonorrhoeae dihydrofolate reductase by a substituted 2,4-diamino-5-benzylpyrimidine

Neisseria gonorrhoeae dihydrofolate reductase undergoes a time-dependent, irreversible inactivation by 2,4-diamino-5-[3,5-dimethoxy-4-(p-bromoacetamidophenoxy)benzyl] pyrimidine. The kinetics of inactivation are consistent with the reversible formation of an enzyme-inhibitor complex followed by covalent binding to the enzyme. The reversible component is competitive with dihydrofolate and has an inhibitor binding constant of 10 nM. Irreversible inactivation proceeds as a pseudo first-order process with a minimum inactivation half-time of 20 min and a Ki of 28 nM. Using radiolabeled inhibitor, it was shown that approximately 1 mol of ligand was covalently bound to the enzyme/mol of methotrexate binding site when the enzyme was completely inhibited. Radiolabeled inhibitor remained associated with the enzyme following denaturation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyanogen bromide cleavage of the 14C-labeled enzyme-inhibitor complex yielded only one radioactive polypeptide, and sequence determinations showed that His-25 was modified by covalent attachment of the inhibitor. When dihydrofolate reductases from Lactobacillus casei, Streptococcus faecium, Escherichia coli, SR-1 rodent lymphoma, and chicken liver were tested with the affinity label, only the L. casei enzyme showed a time-dependent increase in inhibition. These data, along with comparisons of known amino acid sequences and x-ray crystal structures, were used to make predictions concerning the three-dimensional conformation of the gonococcal enzyme.     

Structure and function of alternative proton-relay mutants of dihydrofolate reductase.

Using site-specific mutagenesis, we have constructed two mutants of Escherichia coli dihydrofolate reductase (ecDHFR) to investigate further the function of a weakly acidic side chain at position 27 in substrate protonation: Asp27-->Glu (D27E) and Asp27-->Cys (D27C). The crystal structure of D27E ecDHFR in a binary complex with methotrexate shows that the side-chain oxygen atoms of Glu27 are in almost precisely the same location as those of Asp27 in the wild-type enzyme. Kinetic evidence indicates that Glu27 can indeed function efficiently in the proton relay to dihydrofolate. Even though vertebrate DHFRs all have a glutamic acid at the structurally equivalent position, the kinetic properties of Glu27 ecDHFR more closely resemble those of wild-type bacterial DHFRs than of vertebrate DHFRs. The D27C mutation produced an enzyme still capable of relaying a proton to dihydrofolate, but with the intrinsic pKa in its pH-activity profiles shifted upward to values characteristic of the more basic thiolate group. The crystal structure of the binary complex with methotrexate reveals two unexpected features: (1) the Cys27 sulfhydryl group does not point toward the pteridine-binding site, but the side chain of this residue is instead rotated 120 degrees to interact with a tyrosine side chain projecting from a neighboring beta-strand; (2) a bound ethanol molecule occupies a cavity adjacent to methotrexate. Ethanol is a component of the crystallization medium.     

Affinity labeling of dihydrofolate reductase with an antifolate glyoxal

Dihydrofolate reductases from different species contain several highly conserved arginines, some of which have been shown by x-ray crystallography to have their guanido groups near the p-aminobenzoyl glutamate moiety of enzyme-bound methotrexate. The orientation of one of these (Arg-52) appears to be completely reversed in comparing the crystal structures of Escherichia coli with Lactobacillus casei enzyme (Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., and Kraut, J. (1982). J. Biol. Chem. 257, 13650-13662). We synthesized a novel antifolate containing a glyoxal group designed to react specifically with active-site guanido groups which are able to approach the p-aminobenzoyl carbonyl of methotrexate. The binding of this compound to the enzyme was competitive with dihydrofolate (DHF) in ordinary buffers. In borate buffer at pH 8.0 it inactivated dihydrofolate reductases from both E. coli and L. casei at similar maximum rates, while the chicken liver enzyme was more slowly inactivated. The inactivation was stoichiometric, paralleled the loss of the glyoxal chromophore, and showed saturation kinetics. Inhibitor binding and thus inactivation was enhanced by NADPH, while DHF protected the enzyme. This allowed calculation of the Kd for DHF which was found to be identical with its Km. The stoichiometrically inactivated enzyme displayed the 340-nm chromophore characteristic of 4-aminopteridines bound to dihydrofolate reductase confirming active-site labeling with normal orientation of the ligand. The ligand remained covalently bound to inactivated enzyme upon denaturation at low pH but dissociated at neutral pH. Computer graphic modeling of the crystal structures predicted reaction of Arg-31 but not Arg-52 in L. casei dihydrofolate reductase and of only Arg-52 in the E. coli enzyme. Purification of the CNBr fragments from the inactivated enzymes gave a single labeled peptide for each species. The particular peptide tagged in each case was unaffected by the presence of NADPH and was in excellent agreement with the crystallographic predictions.     

Comparison of solution structures of dihydrofolate reductases and enzyme-ligand complexes using cross-reacting antibodies

Polyclonal antibodies against dihydrofolate reductase (DHFR) from the human lymphoblastoid cell line WIL-2/M4 were used as probes to compare the antigenic structures in solution of native DHFRs obtained from a broad range of species and their complexes with substrate, cofactor, and folate antagonist inhibitors. All these antibodies could bind to the denatured human DHFR, indicating that they were specific for the primary structure of this enzyme. Denatured chicken liver and L1210 murine leukemic DHFRs competed for all of the antibodies that bound to the human enzyme, although less effectively than the denatured human enzyme, showing the presence of similar epitopes among the vertebrate enzymes. However, both direct binding and competition experiments showed low antibody cross-reactivities with native chicken liver (8%) and murine (10%) DHFRs, suggesting differences in the disposition of similar epitopes in these enzymes. The lactobacillus casei DHFR showed a low amount (less than 2%) of cross-reactivity with the antibodies while the same antibodies did not cross-react with the Escherichia coli enzyme. DHFR from soybean seedlings competed for a large proportion (70%) of the anti-human DHFR antibodies, indicating a close similarity in the antigenic structures of plant and animal DHFRs. Binary complexes of the L. casei, avian, murine, and human DHFRs with dihydrofolate, methotrexate (MTX), trimethoprim (TMP), NADPH, and NADP+ all showed significantly lower antibody binding capacity as compared with the corresponding free enzymes. Further, these ligands inhibited antibody binding to the enzyme to varying degrees.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR structures of apo L. casei dihydrofolate reductase and its complexes with trimethoprim and NADPH: contributions to positive cooperative binding from ligand-induced refolding, conformational changes, and interligand hydrophobic interactions

In order to examine the origins of the large positive cooperativity (ΔG(0)(coop) = -2.9 kcal mol(-1)) of trimethoprim (TMP) binding to a bacterial dihydrofolate reductase (DHFR) in the presence of NADPH, we have determined and compared NMR solution structures of L. casei apo DHFR and its binary and ternary complexes with TMP and NADPH and made complementary thermodynamic measurements. The DHFR structures are generally very similar except for the A-B loop region and part of helix B (residues 15-31) which could not be directly detected for L. casei apo DHFR because of line broadening from exchange between folded and unfolded forms. Thermodynamic and NMR measurements suggested that a significant contribution to the cooperativity comes from refolding of apo DHFR on binding the first ligand (up to -0.95 kcals mol(-1) if 80% of A-B loop requires refolding). Comparisons of Cα-Cα distance differences and domain rotation angles between apo DHFR and its complexes indicated that generally similar conformational changes involving domain movements accompany formation of the binary complexes with either TMP or NADPH and that the binary structures are approaching that of the ternary complex as would be expected for positive cooperativity. These favorable ligand-induced structural changes upon binding the first ligand will also contribute significantly to the cooperative binding. A further substantial contribution to cooperative binding results from the proximity of the bound ligands in the ternary complex: this reduces the solvent accessible area of the ligand and provides a favorable entropic hydrophobic contribution (up to -1.4 kcal mol(-1)).     

Moritella cold-active dihydrofolate reductase: are there natural limits to optimization of catalytic efficiency at low temperature?

Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2 degrees C or less. The enzyme is monomeric (Mr=18,291), 55% identical to its Escherichia coli counterpart, and displays Tm and denaturation enthalpy changes much lower than E. coli and Thermotoga maritima homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0 degrees C. At mesophilic temperatures the apparent Km value for dihydrofolate is 50- to 80-fold higher than for E. coli, Lactobacillus casei, and T. maritima dihydrofolate reductases, whereas the apparent Km value for NADPH, though higher, remains in the same order of magnitude. At 5 degrees C these values are not significantly modified. The enzyme is also much less sensitive than its E. coli counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency (kcat/Km) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.     

EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase

Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer. Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase. The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD. In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz. From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted. Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5). To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied. All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD. The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety. Two further hfc's have been detected and assigned to the inequivalent protons at C(1'). Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees.     

Construction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductase

We have explored the substrate protonation mechanism of Escherichia coli dihydrofolate reductase by changing the location of the proton donor. A double mutant was constructed in which the proton donor of the wild-type enzyme, aspartic acid-27, has been changed to serine and simultaneously an alternative proton donor, glutamic acid, has replaced threonine at position 113. The active site of the resulting variant enzyme molecule should therefore somewhat resemble that proposed for the R67 plasmid-encoded dihydrofolate reductase [Matthews, D. A., Smith, S. L., Baccanari, D. P., Burchall, J. J., Oatley, S. J., & Kraut, J. (1986) Biochemistry 25, 4194]. At pH 7, the double-mutant enzyme has a 3-fold greater kcat and an unchanged Km(dihydrofolate) as compared with the single-mutant Asp-27----Ser enzyme described previously [Howell, E. E., Villafranca, J. E., Warren, M. S., Oatley, S. J., & Kraut, J. (1986) Science (Washington, D.C.) 231, 1123]. Additionally, its activity vs pH profiles together with observed deuterium isotope effects, suggest that catalysis depends on an acidic group with a pKa of 8. It is concluded that the dihydropteridine ring of a bound substrate molecule can indeed be protonated by a glutamic acid side chain at position 113 (instead of an aspartic acid side chain at position 27), but with greatly decreased efficiency: at pH 7, the double mutant still has a 25-fold lower kcat (1.2 s-1) and a 2900-fold lower kcat/km(dihydrofolate) (8.6 X 10(3) s-1 M-1) than the wild-type enzyme.     

DFT studies of the disaccharide, alpha-maltose: relaxed isopotential maps

The disaccharide, alpha-maltose, forms the molecular basis for the analysis of the structure of starch, and determining the conformational energy landscape as the molecule oscillates around the glycosidic bonds is of importance. Thus, it is of interest to determine, using density functionals and a medium size basis set, a relaxed isopotential contour map plotted as a function of the phi(H) and psi(H) dihedral angles. The technical aspects include the method of choosing the starting conformations, the choice of scanning step size, the method of constraining the specific dihedral angles, and the fitting of data to obtain well defined contour maps. Maps were calculated at the B3LYP/6-31+G( *) level of theory in 5 degrees intervals around the (phi(H),psi(H))=(0 degrees ,0 degrees ) position, out to approximately +/-30 degrees or greater, for gg-gg'-c, gg-gg'-r, gt-gt'-c, gt-gt'-r, tg-tg'-c, and tg-tg'-r conformers, as well as one-split gg(c)-gg'(r) conformer. The results show that the preferred conformation of alpha-maltose in vacuo depends strongly upon the hydroxyl group orientations ('c'/'r'), but the energy landscape moving away from the minimum-energy position is generally shallow and transitions between conformational positions can occur without the addition of significant energy. Mapped deviations of selected parameters such as the dipole moment; the C1-O1-C4', H1-C1-O1, and H4'-C4'-O1 bond angles; and deviations in hydroxymethyl rotamers, O5-C5-C6-O6, O5'-C5'-C6'-O6', C5-C6-O6-H, and C5'-C6'-O6'-H', are presented. These allow visualization of the structural and energetic changes that occur upon rotation about the glycosidic bonds. Interactions across the bridge are visualized by deviations in H(O2)...O3', H(O3')...O2, and H1...H4' distances and the H(O2)-O2-C2-C1 and H'(O3')-O3'-C3'-C4' hydroxyl dihedral angles.     

Molecular conformation prostaglandin A1 monoclinic crystalline polymorph.

The molecular conformation of the monoclinic crystalline polymorph of prostaglandin A1 has been determined by X-ray diffraction techniques. The space group is P21 with a = 13.637(2), b = 7.567(1), c = 10.576(2) A, beta = 107.37(3) degrees; Dc = 1.073 g.cm-3 for Z = 2. The molecular conformation is characterized by the nearly parallel arrangement of the C1-C7 and C13-C20 side chains, with a general flattening of the overall structure when compared with the orthorhombic polymorph. The cyclopentenone moiety assumes a C8 envelope conformation with C8 and O9 displaced +0.29 A and -0.18 A from the C9-C10=C11-C12 plane respectively. Concerted, small varations of the torsion angles, primarily about the C8-C12, C14-C15 and C16-C17 bonds, bring the monoclinic and orthorhombic conformations into coincidence.     

Trimethoprim induces heat shock proteins and protein aggregation in E. coli cells

Trimethoprim (TMP), an inhibitor of dihydrofolate reductase, decreases the level of tetrahydrofolate supplying one-carbon units for biosynthesis of nucleotides, proteins, and panthotenate. We have demonstrated for the first time that one of the effects of the TMP action in E. coli cells is protein aggregation and induction of heat shock proteins (Hsps). TMP caused induction of DnaK, DnaJ, GroEL, ClpB, and IbpA/B Hsps. Among these Hsps, IbpA/B were most efficiently induced by TMP and coaggregated with the insoluble proteins. Upon folate stress, deletion of the delta ibpA/B operon resulted in increased protein aggregation but did not influence cell viability.     

Analysis of complexes of inhibitors with Cryptosporidium hominis DHFR leads to a new trimethoprim derivative

Cryptosporidiosis, an opportunistic infection affecting immunocompromised patients, the elderly, and children, is still an untreatable disease since the causative agent, Cryptosporidium hominis, is essentially resistant to all clinically used antimicrobial agents. In order to accelerate the design of new potent and selective inhibitors targeting dihydrofolate reductase of C. hominis (ChDHFR), we determined the structural basis for the potency of existing DHFR inhibitors using superpositions of the structure of ChDHFR with other species and analysis of active site complexes of ChDHFR bound to ligands exhibiting a wide range of IC(50) values. This information was used to develop an accurate docking model capable of identifying potent inhibitors in silico. A series of C7-trimethoprim derivatives, designed to exploit a unique pocket in ChDHFR, was synthesized and evaluated; 7-ethyl TMP has four times higher activity than TMP against ChDHFR.     

Iclaprim, a novel diaminopyrimidine with potent activity on trimethoprim sensitive and resistant bacteria

Iclaprim, a new selective dihydrofolate inhibitor was synthesized based on rational drug design. Iclaprim's interaction with a resistant Staphylococcus aureus dihydrofolate reductase (DHFR) is outlined in comparison to trimethoprim (TMP). This compound is active against methicillin, TMP and vancomycin resistant strains. Arpida Ltd. is developing Iclaprim for serious hospital infections from Gram-positive pathogens and respiratory tract infections.     

Trimethoprim-induced elevation of dihydrofolate reductase activity in human leukocytes.

The administration of trimethoprim (TMP)--a diamino benzylpyrimidine compound which binds very tightly the bacterial dihydrofolate reductase--was accompanied by the appearance of measurable levels of dihydrofolate reductase in peripheral leukocytes from patients with nonhematological diseases. In all instances, enzyme activity rose rapidly between the fourth and eighth day after TMP. The time course of the rise and fall of dihydrofolate activity approaches cellular life span and is similar to that obtained after methotrexate or triamterene administration. Dihydrofolate reductases, partially purified from leukocytes of patients treated with TMP, bone marrow and leukemic leukocytes, had simila molecular weights, pH optima, Ki of inhibitor (methotrexate); they were stimulated to the same degree by KCl and urea. Electrophoresis of the enzyme on cellulose acetate strip resulted in the separation of two enzymatically active protein components. No differences in the electrophoretic behavior of the three blood cell enzymes were noted. The findings noted above are consistent with the suggestion that the observed rise in dihydrofolate reductase activity is a quantitative one. Moreover, the effect of TMP in vivo is discussed in comparison with the currently held hypothesis for methotrexate action (stabilization by the drug of a previously synthetized enzyme).     

Effects of pyrimidine salvage inhibitors on uracil incorporation of Toxoplasma gondii

Metabolic inhibitors which act in the process of pyrimidine salvage influenced on the uracil incorporation into nucleic acids of Toxoplasma. Inhibitors of dihydrofolate reductase, pyrimethamine and methotrexate, and inhibitors of thymidylate synthase, fluoro-uridine, fluoro-dUMP and fluoro-uracil, diminished isotopic uracil uptake in dose-dependent manners. Azauridine which suppresses de novo pyrimidine biosynthesis did not affect the salvage even in a relatively high dose. These results suggested that the activation of uracil salvage should be closely related with the function of TMP biosynthetic enzymes. The pattern of thymidine uptake had no differences between control HL-60 cells and Toxoplasma infected cells, which did not reflect the specific proliferation of Toxoplasma. It can be exploited to characterize the effects of various compounds related with the proliferation of Toxoplasma, especially its DNA synthesis.     

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.     

Crystal Structures of Wild-type and Mutant Methicillin-resistant Staphylococcus aureus Dihydrofolate Reductase Reveal an Alternate Conformation of NADPH That May Be Linked to Trimethoprim Resistance

Both hospital- and community-acquired Staphylococcus aureus infections have become major health concerns in terms of morbidity, suffering and cost. Trimethoprim-sulfamethoxazole (TMP-SMZ) is an alternative treatment for methicillin-resistant S. aureus (MRSA) infections. However, TMP-resistant strains have arisen with point mutations in dihydrofolate reductase (DHFR), the target for TMP. A single point mutation, F98Y, has been shown biochemically to confer the majority of this resistance to TMP. Using a structure-based approach, we have designed a series of novel propargyl-linked DHFR inhibitors that are active against several trimethoprim-resistant enzymes. We screened this series against wild-type and mutant (F98Y) S. aureus DHFR and found that several are active against both enzymes and specifically that the meta-biphenyl class of these inhibitors is the most potent. In order to understand the structural basis of this potency, we determined eight high-resolution crystal structures: four each of the wild-type and mutant DHFR enzymes bound to various propargyl-linked DHFR inhibitors. In addition to explaining the structure-activity relationships, several of the structures reveal a novel conformation for the cofactor, NADPH. In this new conformation that is predominantly associated with the mutant enzyme, the nicotinamide ring is displaced from its conserved location and three water molecules complete a network of hydrogen bonds between the nicotinamide ring and the protein. In this new position, NADPH has reduced interactions with the inhibitor. An equilibrium between the two conformations of NADPH, implied by their occupancies in the eight crystal structures, is influenced both by the ligand and the F98Y mutation. The mutation induced equilibrium between two NADPH-binding conformations may contribute to decrease TMP binding and thus may be responsible for TMP resistance.