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.     

Role of S65, Q67, I68, and Y69 residues in homotetrameric R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) shares no sequence or structural homology with chromosomal DHFRs. This enzyme arose recently in response to the clinical use of the antibacterial drug trimethoprim. R67 DHFR is a homotetramer possessing a single active site pore. A high-resolution crystal structure shows the homotetramer possesses exact 222 symmetry [Narayana, N., et al. (1995) Nat. Struct. Biol. 2, 1018-1025]. This symmetry dictates four symmetry-related binding sites must exist for each substrate as well as each cofactor. Isothermal titration calorimetry studies, however, indicate only two molecules bind: either two dihydrofolate molecules, two NADPH molecules, or one substrate and one cofactor [Bradrick, T. D., et al. (1996) Biochemistry 35, 11414-11424]. The latter is the productive ternary complex. To evaluate the role of S65, Q67, I68, and Y69 residues, located near the center of the active site pore, site-directed mutagenesis was performed. One mutation in the gene creates four mutations per active site pore which typically result in large cumulative effects. Steady state kinetic data indicate the mutants have altered K(m) values for both cofactor and substrate. For example, the Y69F R67 DHFR displays an 8-fold increase in the K(m) for dihydrofolate and a 20-fold increase in the K(m) for NADPH. Residues involved in ligand binding in R67 DHFR display very little, if any, specificity, consistent with their possessing dual roles in binding. These results support a model where R67 DHFR utilizes an unusual "hot spot" binding surface capable of binding both ligands and indicate this enzyme has adopted a novel yet simple approach to catalysis.     

Calorimetric studies of ligand binding in R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) is a novel bacterial protein that possesses 222 symmetry and a single active site pore. Although the 222 symmetry implies that four symmetry-related binding sites must exist for each substrate as well as for each cofactor, various studies indicate only two molecules bind. Three possible combinations include two dihydrofolate molecules, two NADPH molecules, or one substrate plus one cofactor. The latter is the productive ternary complex. To explore the role of various ligand substituents during binding, numerous analogues, inhibitors, and fragments of NADPH and/or folate were used in both isothermal titration calorimetry (ITC) and K(i) studies. Not surprisingly, as the length of the molecule is shortened, affinity is lost, indicating that ligand connectivity is important in binding. The observed enthalpy change in ITC measurements arises from all components involved in the binding process, including proton uptake. As a buffer dependence for binding of folate was observed, this likely correlates with perturbation of the bound N3 pK(a), such that a neutral pteridine ring is preferred for pairwise interaction with the protein. Of interest, there is no enthalpic signal for binding of folate fragments such as dihydrobiopterin where the p-aminobenzoylglutamate tail has been removed, pointing to the tail as providing most of the enthalpic signal. For binding of NADPH and its analogues, the nicotinamide carboxamide is quite important. Differences between binary (binding of two identical ligands) and ternary complex formation are observed, indicating interligand pairing preferences. For example, while aminopterin and methotrexate both form binary complexes, albeit weakly, neither readily forms ternary complexes with the cofactor. These observations suggest a role for the O4 atom of folate in a pairing preference with NADPH, which ultimately facilitates catalysis.     

Crystal structure of a type II dihydrofolate reductase catalytic ternary complex

Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR.DHF.NADP+ catalytic complex, resolved to 1.26 A. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine ring. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67'-Y69' residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationally challenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation.     

Insights into the sirtuin mechanism from ternary complexes containing NAD+ and acetylated peptide

Sirtuin proteins comprise a unique class of NAD+-dependent protein deacetylases. Although several structures of sirtuins have been determined, the mechanism by which NAD+ cleavage occurs has remained unclear. We report the structures of ternary complexes containing NAD+ and acetylated peptide bound to the bacterial sirtuin Sir2Tm and to a catalytic mutant (Sir2Tm(H116Y)). NAD+ in these structures binds in a conformation different from that seen in previous structures, exposing the alpha face of the nicotinamide ribose to the carbonyl oxygen of the acetyl lysine substrate. The NAD+ conformation is identical in both structures, suggesting that proper coenzyme orientation is not dependent on contacts with the catalytic histidine. We also present the structure of Sir2Tm(H116A) bound to deacteylated peptide and 3'-O-acetyl ADP ribose. Taken together, these structures suggest a mechanism for nicotinamide cleavage in which an invariant phenylalanine plays a central role in promoting formation of the O-alkylamidate reaction intermediate and preventing nicotinamide exchange.     

Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67

Crystalline R67 dihydrofolate reductase (DHFR) is a dimeric molecule with two identical 78 amino acid subunits, each folded into a beta-barrel conformation. The outer surfaces of the three longest beta strands in each protomer together form a third beta barrel having six strands at the subunit interface. A unique feature of the enzyme structure is that while the intersubunit beta barrel is quite regular over most of its surface, an 8-A "gap" runs the full length of the barrel, disrupting potential hydrogen bonds between beta-strand D in subunit I and the adjacent corresponding strand of subunit II. It is proposed that this deep groove is the NADPH binding site and that the association between protein and cofactor is modulated by hydrogen-bonding interactions along one face of this antiparallel beta-barrel structure. A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase. Geometrical comparison of this model with an experimentally determined structure for chicken DHFR suggests that chromosomal and type II R-plasmid specified enzymes may have independently evolved similar catalytic machinery for substrate reduction.     

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.     

Conformation of NADP+ bound to a type II dihydrofolate reductase

Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are sequence and structurally different from known chromosomal DHFRs. These plasmid-derived DHFRs are responsible for confering trimethoprim resistance to the host strain. A derivative of R388 DHFR, RBG200, has been cloned and its physical properties have been characterized. This enzyme has been shown to transfer the pro-R hydrogen of NADPH to its substrate, dihydrofolate, making it a member of the A-stereospecific class of dehydrogenases [Brito, R. M. M., Reddick, R., Bennett, G. N., Rudolph, F. B., & Rosevear, P. R. (1990) Biochemistry 29,9825]. Two distinct binary RBG200.NADP+ complexes were detected. Addition of NADP+ to RBG200 DHFR results in formation of an initial binary complex, conformation I, which slowly interconverts to a second more stable binary complex, conformation II. The binding of NADP+ to RBG200 DHFR in the second binary complex was found to be weak, KD = 1.9 +/- 0.4 mM. Transferred NOEs were used to determine the conformation of NADP+ bound to RBG200 DHFR. The initial slope of the NOE buildup curves, measured from the intensity of the cross-peaks as a function of the mixing time in NOESY spectra, allowed interproton distances on enzyme-bound NADP+ to be estimated. The experimentally measured distances were used to define upper and lower bound distance constraints between proton pairs in distance geometry calculations. All NADP+ structures consistent with the experimental distance bounds were found to have a syn conformation about the nicotinamide-ribose (X = 94 +/- 26 degrees) and an anti conformation about the adenine-ribose (X = -92 +/- 32 degrees) glycosidic bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

A balancing act between net uptake of water during dihydrofolate binding and net release of water upon NADPH binding in R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH.DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR.     

Effects of single-tryptophan mutations on R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers clinical resistance to the antibacterial drug trimethoprim. This enzyme shows no sequence or structural homology to the chromosomal DHFRs. The active form of the protein is a homotetramer possessing D(2) symmetry and a single active-site pore. Two tryptophans occur per monomer: W38 and its symmetry-related residues (W138, W238, and W338) occur at the dimer-dimer interfaces, while W45 and its symmetry-related partners (W145, W245, and W345) occur at the monomer-monomer interfaces. Two single-tryptophan mutant genes were constructed to determine the structural and functional consequences of four mutations per tetramer. The W45F mutant retains full enzyme activity and the fluorescence environment of the unmutated W38 residues clearly monitors ligand binding and a pH dependent tetramer right harpoon over left harpoon 2 dimers equilibrium. In contrast, four simultaneous W38F mutations at the dimer-dimer interfaces result in tetramer destabilization. The ensuing dimer is relatively inactive, as is dimeric wild-type R67 DHFR. A comparison of emission spectra indicates the fluorescent signal of wild-type R67 DHFR is dominated by the contribution from W38. Equilibrium unfolding/folding curves at pH 5.0, where all protein variants are dimeric, indicate the environment monitored by the W38 residue is slightly less stable than the environment monitored by the W45 residue.     

Breaking symmetry: mutations engineered into R67 dihydrofolate reductase,a D2 symmetric homotetramer possessing a single active site pore

R67 dihydrofolate reductase (DHFR) is an enzyme, encoded by an R-plasmid, that confers resistance to the antibacterial agent trimethoprim. This homotetramer possesses a single active site pore and exact 222 symmetry. The symmetry imposes constraints on the ability of the enzyme to optimize binding of the substrate, dihydrofolate (DHF), and the cofactor, NADPH, resulting in a "one site fits both ligands" approach. This approach allows formation of either a NADPH.NADPH, dihydrofolate.dihydrofolate, or NADPH.dihydrofolate complex. The first two complexes are nonproductive, while the third is the productive catalytic species. To break the symmetry of the active site, a tandem array of four R67 DHFR genes has been linked in frame, allowing individual manipulation of each gene copy. Various numbers and combinations of asymmetric Q67H mutations have been engineered into the tandem gene array. The Q67H mutation was chosen for investigation as it was previously found to tighten binding to both dihydrofolate and NADPH by approximately 100-fold in homotetrameric R67 DHFR [Park, H., Bradrick, T. D., and Howell, E. E. (1997) Protein Eng. 10, 1415-1424]. Nonadditive effects on ligand binding are observed when one to four mutations are inserted, indicating either conformational changes in the protein or different cooperativity patterns in the ligand-ligand interactions. From steady state kinetics, addition of Q67H mutations does not drastically affect formation of the NADPH.dihydrofolate complex; however, a large energy difference between the productive and nonproductive complexes is no longer maintained. A role for Q67 in discriminating between these various states is proposed. Since theories of protein evolution suggest gene duplication followed by accumulation of mutations can lead to divergence of activity, this study is a first step toward asking if introduction of asymmetric mutations in the quadrupled R67 DHFR gene can lead to optimization of ligand binding sites.     

Defining the binding site of homotetrameric R67 dihydrofolate reductase and correlating binding enthalpy with catalysis

R67 dihydrofolate reductase (DHFR) is a novel protein that possesses 222 symmetry. A single active site pore traverses the length of the homotetramer. Although the 222 symmetry implies that four symmetry-related binding sites should exist for each substrate as well as each cofactor, isothermal titration calorimetry (ITC) studies indicate only two molecules bind. Three possible combinations include two dihydrofolate molecules, two NADPH molecules, or one substrate with one cofactor. The latter is the productive ternary complex. To evaluate the roles of A36, Y46, T51, G64, and V66 residues in binding and catalysis, a site-directed mutagenesis approach was employed. One mutation per gene produces four mutations per active site pore, which often result in large cumulative effects. Conservative mutations at these positions either eliminate the ability of the gene to confer trimethoprim resistance or have no effect on catalysis. This result, in conjunction with previous mutagenesis studies on K32, K33, S65, Q67, I68, and Y69 [Strader, M. B., et al. (2001) Biochemistry 40, 11344-11352; Hicks, S. N., et al. (2003) Biochemistry 42, 10569-10578; Park, H., et al. (1997) Protein Eng. 10, 1415-1424], allows mapping of the active site surface. Residues for which conservative mutations have large effects on binding and catalysis include K32, Q67, I68, and Y69. These residues form a stripe that establishes the ligand binding surface. Residues that accommodate conservative mutations that do not greatly affect catalysis include K33, Y46, T51, S65, and V66. Isothermal titration calorimetry studies were also conducted on many of the mutants described above to determine the enthalpy of folate binding to the R67 DHFR.NADPH complex. A linear correlation between this DeltaH value and log k(cat)/K(m) is observed. Since structural tightness appears to be correlated with the exothermicity of the binding interaction, this leads to the hypothesis that enthalpy-driven formation of the ternary complex in these R67 DHFR variants plays a strong role in catalysis. Use of the alternate cofactor, NADH, extends this correlation, indicating preorganization of the ternary complex determines the efficiency of the reaction. This hypothesis is consistent with data suggesting R67 DHFR uses an endo transition state (where the nicotinamide ring of cofactor overlaps the more bulky side of the substrate's pteridine ring).     

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.     

Vibrational structure of dihydrofolate bound to R67 dihydrofolate reductase.

R67 is a Type II dihydrofolate reductase (DHFR) that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate by facilitating the addition of a proton to N5 of DHF and the transfer of a hydride ion from NADPH to C6. Because this enzyme is a plasmid-encoded DHFR from trimethoprim-resistant bacteria, extensive studies on R67 with various methods have been performed to elucidate its reaction mechanism. Here, Raman difference measurements, conducted on the ternary complex of R67.NADP(+).DHF believed to be an accurate mimic of the productive DHFR.NADPH.DHF complex, show that the pK(a) of N5 in the complex is less than 4. This is in clear contrast to the behavior observed in Escherichia coli DHFR, a substantially more efficient enzyme, where the pK(a) of bound DHF at N5 is increased to 6.5 compared with its solution value of 2.6. A comparison of the ternary complexes in R67 and E. coli DHFRs suggests that enzymic raising of the pK(a) at N5 can significantly increase the catalytic efficiency of the hydride transfer step. However, R67 shows that even without such a strategy an effective DHFR can still be designed.     

Mechanism of coenzyme recognition and binding revealed by crystal structure analysis of ferredoxin-NADP+ reductase complexed with NADP+

The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyses the production of NADPH in photosynthesis. The three-dimensional structure of FNR presents two distinct domains, one for binding of the FAD prosthetic group and the other for NADP+ binding. In spite of extensive experiments and different crystallographic approaches, many aspects about how the NADP+ substrate binds to FNR and how the hydride ion is transferred from FAD to NADP+ remain unclear. The structure of an FNR:NADP+ complex from Anabaena has been determined by X-ray diffraction analysis of the cocrystallised units to 2.1 A resolution. Structural perturbation of FNR induced by complex formation produces a narrower cavity in which the 2'-phospho-AMP and pyrophosphate portions of the NADP+ are perfectly bound. In addition, the nicotinamide mononucleotide moiety is placed in a new pocket created near the FAD cofactor with the ribose being in a tight conformation. The crystal structure of this FNR:NADP+ complex obtained by cocrystallisation displays NADP+ in an unusual conformation and can be considered as an intermediate state in the process of coenzyme recognition and binding. Structural analysis and comparison with previously reported complexes allow us to postulate a mechanism which would permit efficient hydride transfer to occur. Besides, this structure gives new insights into the postulated formation of the ferredoxin:FNR:NADP+ ternary complex by prediction of new intermolecular interactions, which could only exist after FNR:NADP+ complex formation. Finally, structural comparison with the members of the broad FNR structural family also provides an explanation for the high specificity exhibited by FNR for NADP+/H versus NAD+/H.     

Role of ionic interactions in ligand binding and catalysis of R67 dihydrofolate reductase

R67 dihydrofolate reductase (DHFR), which catalyzes the NADPH dependent reduction of dihydrofolate to tetrahydrofolate, belongs to a type II family of R-plasmid encoded DHFRs that confer resistance to the antibacterial drug trimethoprim. Crystal structure data reveals this enzyme is a homotetramer that possesses a single active site pore. Only two charged residues in each monomer are located near the pore, K32 and K33. Site-directed mutants were constructed to probe the role of these residues in ligand binding and/or catalysis. As a result of the 222 symmetry of this enzyme, mutagenesis of one residue results in modification at four related sites. All mutants at K32 affected the quaternary structure, producing an inactive dimer. The K33M mutant shows only a 2-4-fold effect on K(m) values. Salt effects on ligand binding and catalysis for K33M and wildtype R67 DHFRs were investigated to determine if these lysines are involved in forming ionic interactions with the negatively charged substrates, dihydrofolate (overall charge of -2) and NADPH (overall charge of -3). Binding studies indicate that two ionic interactions occur between NADPH and R67 DHFR. In contrast, the binding of folate, a poor substrate, to R67 DHFR.NADPH appears weak as a titration in enthalpy is lost at low ionic strength. Steady-state kinetic studies for both wild type (wt) and K33M R67 DHFRs also support a strong electrostatic interaction between NADPH and the enzyme. Interestingly, quantitation of the observed salt effects by measuring the slopes of the log of ionic strength versus the log of k(cat)/K(m) plots indicates that only one ionic interaction is involved in forming the transition state. These data support a model where two ionic interactions are formed between NADPH and symmetry related K32 residues in the ground state. To reach the transition state, an ionic interaction between K32 and the pyrophosphate bridge is broken. This unusual scenario likely arises from the constraints imposed by the 222 symmetry of 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.     

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.     

Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions

Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such “half-native” tetramers were shown to procure native-like catalytic activity, with similar K_M values but k_cat values 5- to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (T_m ∼12°C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active-site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation.     

The Influence of Starting Coordinates in Free Energy Simulations of Ligand Binding to Dihydrofolate Reductase

Abstract

Molecular dynamics (MD) simulation combined with free energy perturbation (FEP) methods have been used to study the key structural differences and relative free energies for the binding of 6-methyl-N5-deazapterin (N8 protonated) and the 8-substituted compound, 6,8-dimethyl-N5-deazapterin (N3 protonated), to dihydrofolate reductase (DHFR). The free energy changes have been calculated using a variety of initial X-ray coordinates derived from bacterial and vertebrate (including human) DHFRs, and both with and without the reduced cofactor nicotinamide adenine dinucleotide (NADPH) bound. Given a sufficiently long simulation time for the FEP calculations (ca. 200 ps), all structures obtained after mutating 6,8-methyl-N5-deazapterin to 6-methyl-N5-deazapterin exhibited hydrogen bond formation between a backbone carbonyl group of DHFR and H(N8) of 6-methyl-N5-deazapterin, analogous to that found in the X-ray crystal structure of N5-deazafolate(N8 protonated) bound to human DHFR. However, both simulation and experiment suggest this additional H-bonding does not greatly enhance thermodynamic stability, with experiment indicating at most a factor of 2 difference in the relative affinities of the two ligand cations for vertebrate DHFR. Moreover, a binding differential of 10 in favour of the protonated 8-substituted compound is found experimentally for bacterial DHFR. The MD/FEP calculations suggest that the relative cost of ligand desolvation may largely cancel the lowering of free energy obtained in the active site, resulting in predicted binding differences within the range indicated by the vertebrate and bacterial DHFR experiments. However, the theoretical free energy changes could not be obtained with the accuracy required for the rationalization of the observed species dependence. While sampling difficulties are known to be inherent in MD simulation methodologies, these studies with several initial coordinate sets have demonstrated the contribution of coordinate choice to this problem. The results indicate that for demanding protein-ligand binding problems such as this one, the accuracy of the method may be no better than ± 2 kcal/mol.