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.     

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.     

Role of Lys-32 residues in R67 dihydrofolate reductase probed by asymmetric mutations

R67 dihydrofolate reductase (R67 DHFR) is a novel protein encoded by an R-plasmid that confers resistance to the antibiotic, trimethoprim. This homotetrameric enzyme possesses 222 symmetry, which imposes numerous constraints on the single active site pore, including a "one-site-fits-both" strategy for binding its ligands, dihydrofolate (DHF) and NADPH. Previous studies uncovered salt effects on binding and catalysis (Hicks, S. N., Smiley, R. D., Hamilton, J. B., and Howell, E. E. (2003) Biochemistry 42, 10569-10578), however the one or more residues that participate in ionic contacts with the negatively charged tail of DHF as well as the phosphate groups in NADPH were not identified. Several studies predict that Lys-32 residues were involved, however mutations at this residue destabilize the R67 DHFR homotetramer. To study the role of Lys-32 in binding and catalysis, asymmetric K32M mutations have been utilized. To create asymmetry, individual mutations were added to a tandem array of four in-frame gene copies. These studies show one K32M mutation is tolerated quite well, whereas addition of two mutations has variable effects. Two double mutants, K32M:1+2 and K32M: 1+4, which place the mutations on opposite sides of the pore, reduce kcat. However a third double mutant, K32M: 1+3, that places two mutations on the same half pore, enhances kcat 4- to 5-fold compared with the parent enzyme, albeit at the expense of weaker binding of ligands. Because the kcat/Km values for this double mutant series are similar, these mutations appear to have uncovered some degree of non-productive binding. This non-productive binding mode likely arises from formation of an ionic interaction that must be broken to allow access to the transition state. The K32M:1+3 mutant data suggest this interaction is an ionic interaction between Lys-32 and the charged tail of dihydrofolate. This unusual catalytic scenario arises from the 222 symmetry imposed on the single active site pore.     

Probing the roles of conserved arginine-44 of Escherichia coli dihydrofolate reductase in its function and stability by systematic sequence perturbation analysis

Sequence perturbation analysis is a powerful method to reveal roles of an amino acid residue in function and stability of a protein. By using and improving this method, we studied roles of highly conserved Arg44 of Escherichia coli dihydrofolate reductase (DHFR) in its function and stability. Here, we introduced systematic amino acid substitutions at this position and found that all 19 kinds of amino acid substitutions were tolerated, but the mutations significantly reduced the enzymatic activity and the binding affinity toward the cofactor NADPH. Moreover, the mutational effects on the cofactor binding affinity were well correlated with those on the catalytic activity, indicating that the R44X mutations affect the catalytic activity mainly by modulating the cofactor binding affinity. On the other hand, thermal denaturation measurements showed that most mutations stabilized the protein. Comparison between the mutational effects and various amino acid indices taken from the AAindex database indicated that hydrophobicity and polarity are key determinants of amino acids favorable at this position. These results suggest that through electrostatic interactions Arg44 plays a functional role in retaining the cofactor binding affinity at the cost of the protein stability.     

Functional role for Tyr 31 in the catalytic cycle of chicken dihydrofolate reductase

Despite much work, many key aspects of the mechanism of the dihydrofolate reductase (DHFR) catalyzed reduction of dihydrofolate remain unresolved. In bacterial forms of DHFR both substrate and water access to the active site are controlled by the conformation of the mobile M20 loop. In vertebrate DHFRs only one conformation of the residues corresponding to the M20 loop has been observed. Access to the active site was proposed to be controlled by residue 31. MD simulations of chicken DHFR complexed with substrates and cofactor revealed a closing of the side chain of Tyr 31 over the active site on binding of dihydrofolate. This conformational change was dependent on the presence of glutamate on the para-aminobenzoylamide moiety of dihydrofolate. In its absence, the conformation remained open. Although water could enter the active site and hydrogen bond to N5 of dihydrofolate, indicating the feasibility of water as the proton donor, this was not controlled by the conformation of Tyr 31. The water accessibility of the active site was low for both conformations of Tyr 31. However, when hydride was transferred from NADPH to C6 of dihydrofolate before protonation, the average time during which water was found in hydrogen bonding distance to N5 of dihydrofolate in the active site increased almost fivefold. These results indicated that water can serve as the Broensted acid for the protonation of N5 of dihydrofolate during the DHFR catalyzed reduction.     

Gammaherpesviruses encode functional dihydrofolate reductase activity

We overexpressed and purified from Escherichia coli the dihydrofolate reductase (DHFR) of the gammaherpesviruses human herpesvirus 8 (HHV-8), herpesvirus saimiri (HVS), and rhesus rhadinovirus (RRV). All three enzymes proved catalytically active. The K(m) value of HHV-8 DHFR for dihydrofolate (DHF) was 2.02+/-0.44 microM, that of HVS DHFR was 4.31+/-0.56 microM, and that of RRV DHFR is 7.09+/-0.11 microM. These values are approximately 5-15-fold higher than the K(m) value reported for the human DHFR. The K(m) value of HHV-8 DHFR for NADPH was 1.31+/-0.23 microM, that of HVS DHFR was 3.78+/-0.61 microM, and that of RRV DHFR was 7.47+/-0.59 microM. These values are similar or slightly higher than the corresponding K(m) value of the human enzyme. Methotrexate, aminopterin, trimethoprim, pyrimethamine, and N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), all well-known folate antagonists, inhibited the DHFR activity of the three gammaherpesviruses competitively with respect to DHF but proved markedly less inhibitory to the viral than towards the human enzyme.     

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.     

Site-directed deletion mutants of a carboxyl-terminal region of human dihydrofolate reductase.

Substrate and inhibitor binding to dihydrofolate reductase (DHFR) primarily involves residues in the amino-terminal half of the enzyme; however, antibody binding studies performed in this laboratory suggested that the loop region located in the carboxyl terminus of human DHFR (hDHFR; residues 140-186) is involved in conformational changes that occur upon ligand binding and affect enzyme function (Ratnam, M., Tan, X., Prendergast, N.J., Smith, P.L. & Freisheim, J.H. (1988) Biochemistry 27, 4800-4804). To investigate this observation further, site-directed mutagenesis was used to construct deletion mutants of hDHFR missing 1 (del-1), 2 (del-2), 4 (del-4), and 6 (del-6) residues from loops in the carboxyl terminus of the enzyme. The del-1 mutant enzyme has a two-amino acid substitution in addition to the one-amino acid deletion. Deletion of only one amino acid resulted in a 35% decrease in the specific activity of the enzyme. The del-6 mutant enzyme was inactive. Surprisingly, the del-4 mutant enzyme retained a specific activity almost 33% that of the wild type. The specific activity of the del-2 mutant enzyme was slightly higher (38% wild-type activity) than that of the del-4 mutant. All three active deletion mutants were much less stable than the wild-type enzyme, and all three showed at least a 10-fold increase in Km values for both substrates. The del-1 and del-2 mutants exhibited a similar increase in KD values for both substrate and cofactor. The three active deletion mutants lost activity at concentrations of activating agents such as KCl, urea, and p-hydroxymercuribenzoate that continued to stimulate the wild-type enzyme. Antibody binding studies revealed conformational differences between the wild-type and mutant enzymes both in the absence and presence of bound folate. Thus, although the loops near the carboxyl terminus are far removed from the active site, small deletions of this region significantly affect DHFR function, indicating that the loop structure in mammalian DHFR plays an important functional role in its conformation and catalysis.     

Invariant amino acid replacement affects the dihydrofolate reductase function and its gene expression

Three different forms of dihydrofolate reductase (DHFR) from Escherichia coli with amino acid replacements Thr35----Asp, Asn37----Ser and Arg57----His, and one form containing all three of these changes were obtained by oligonucleotide-directed mutagenesis. These amino acids are on the surface of the protein and two of them (Thr35 and Arg57) are invariant for known sequences of DHFR. Conversion of Asn37----Ser has no effect on the functional activity or the protein level in the cells. The Thr35----Asp replacement leads to a sharp decrease in the protein level, while the addition of a DHFR inhibitor, trimethoprim (Tmp), to the growth medium increases the level of DHFR in the cells. There is a very small quantity of DHFR with all three amino acid changes. The addition of Tmp to the growth medium also leads to an increase in the mutant protein levels. The mutant with the Arg57----His replacement renders the cells sensitive to Tmp, but the level of DHFR is the same as for the wild-type protein. It is suggested that the invariant Thr35 is important for the stable conformation of DHFR whereas Arg57 is essential for protein activity. Various structural and functional aspects of these results are discussed.     

Sequential conformation changes of chinese hamster ovary dihydrofolate reductase at its active sites

The local fluorescence probes, 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS) and NADPH were employed to detect urea-induced conformation changes at each active site of dihydrofolate reductase (DHFR), respectively. The results indicate that local conformation change at DHF/TNS could be superimposed by the conformation change calculated from the enzyme activity change with a three-state model; while at NADPH site it is lagged in the first transition. This difference is further supported by the different relative changes of Michaelis constants at 0, 1 and 1.8 M urea for each substrate. Our results suggest that local conformation at DHF site is more flexible than that at NADPH site, and the urea-induced unfolding could be ascribed to a four-state transition.     

Structure of the Q67H mutant of R67 dihydrofolate reductase-NADP+ complex reveals a novel cofactor binding mode

Plasmid-encoded bacterial R67 dihydrofolate reductase (DHFR) is a NADPH-dependent enzyme unrelated to chromosomal DHFR in amino acid sequence and structure. R67 DHFR is insensitive to the bacterial drug trimethoprim in contrast to chromosomal DHFR. The crystal structure of Q67H mutant of R67 DHFR bound to NADP(+) has been determined at 1.15 angstroms resolution. The cofactor assumes an extended conformation with the nicotinamide ring bound near the center of the active site pore, the ribose and pyrophosphate group (PP(i)) extending toward the outer pore. The ribonicotinamide exhibits anti conformation as in chromosomal DHFR complexes. The relative orientation between the PP(i) and the nicotinamide ribose differs from that observed in chromosomal DHFR-NADP(+) complexes. The coenzyme displays symmetrical binding mode with several water-mediated hydrogen bonds with the protein besides ionic, stacking, and van der Waals interactions. The structure provides a molecular basis for the observed stoichiometry and cooperativity in ligand binding. The ternary model based on the present structure and the previous R67 DHFR-folate complex provides insight into the catalytic mechanism and indicates that the relative orientation of the reactants in plasmid DHFR is different from that seen in chromosomal DHFRs.     

Analysis of hydride transfer and cofactor fluorescence decay in mutants of dihydrofolate reductase: possible evidence for participation of enzyme molecular motions in catalysis.

A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.     

Effects of temperature and viscosity on R67 dihydrofolate reductase catalysis

R67 dihydrofolate reductase (DHFR) is a novel homotetrameric protein that possesses 222 symmetry and a single, voluminous active site pore. This symmetry poses numerous limitations on catalysis; for example, two dihydrofolate (DHF) molecules or two NADPH molecules, or one substrate plus one cofactor can bind. Only the latter combination leads to catalysis. To garner additional information on how this enzyme facilitates transition-state formation, the temperature dependence of binding and catalysis was monitored. The binding of NADPH and DHF is enthalpy-driven. Previous primary isotope effect studies indicate hydride transfer is at least partially rate-determining. Accordingly, the activation energy associated with transition-state formation was measured and is found to be 6.9 kcal/mol (DeltaH(++)(25) = 6.3 kcal/mol). A large entropic component is also found associated with catalysis, TDeltaS(++)(25) = -11.3 kcal/mol. The poor substrate, dihydropteroate, binds more weakly than dihydrofolate (DeltaDeltaG = 1.4 kcal/mol) and displays a large loss in the binding enthalpy value (DeltaDeltaH = 3.8 kcal/mol). The k(cat) value for dihydropteroate reduction is decreased 1600-fold compared to DHF usage. This effect appears to derive mostly from the DeltaDeltaH difference in binding, demonstrating that the glutamate tail is important for catalysis. This result is surprising, as the para-aminobenzoyl-glutamate tail of DHF has been previously shown to be disordered by both NMR and crystallography studies. Viscosity studies were also performed and confirmed that the hydride transfer rate is not sensitive to sucrose addition. Surprisingly, binding of DHF, by both K(m) and K(d) determination, was found to be sensitive to added viscogens, suggesting a role for water in DHF binding.     

Conformation coupled enzyme catalysis: single-molecule and transient kinetics investigation of dihydrofolate reductase

Ensemble kinetics and single-molecule fluorescence microscopy were used to study conformational transitions associated with enzyme catalysis by dihydrofolate reductase (DHFR). The active site loop of DHFR was labeled with a fluorescence quencher, QSY35, at amino acid position 17, and the fluorescent probe, Alexa555, at amino acid 37, by introducing cysteines at these sites with site-specific mutagenesis. The distance between the probes was such that approximately 50% fluorescence resonance energy transfer (FRET) occurred. The double-labeled enzyme retained essentially full catalytic activity, and stopped-flow studies of both the forward and reverse reactions revealed that the distance between probes increased prior to hydride transfer. A fluctuation in fluorescence intensity of single molecules of DHFR was observed in an equilibrium mixture of substrates but not in their absence. Ensemble rate constants were derived from the distributions of lifetimes observed and attributed to a reversible conformational change. Studies were carried out with both NADPH and NADPD as substrates, with no measurable isotope effect. Similar studies with a G121V mutant DHFR resulted in smaller rate constants. This mutant DHFR has reduced catalytic activity, so that the collective data for the conformational change suggest that the conformational change being observed is associated with catalysis and probably represents a conformational change prior to hydride transfer. If the change in fluorescence is attributed to a change in FRET, the distance change associated with the conformational change is approximately 1-2 A. These results are correlated with other measurements related to conformation coupled catalysis.     

R67, the other dihydrofolate reductase: rational design of an alternate active site configuration

R67 dihydrofolate reductase (DHFR) bears no sequence or structural homologies with chromosomal DHFRs. The gene for this enzyme produces subunits that are 78 amino acids long, which assemble into a homotetramer possessing 222 symmetry. More recently, a tandem array of four gene copies linked in-frame was constructed, which produces a monomer containing 312 amino acids named Quad3. Asymmetric mutations in Quad3 have also been constructed to probe the role of Q67 and K32 residues in catalysis. This present study mixes and matches mutations to determine if the Q67H mutation, which tightens binding approximately 100-fold to both dihydrofolate (DHF) and NADPH, can help rescue the K32M mutation. While the latter mutation weakens DHF binding over 60-fold, it concurrently increases kcat by a factor of 5. Two Q67H mutations were added to gene copies 1 and 4 in conjunction with the K32M mutation in gene copies 1 and 3. Addition of these Q67H mutations tightens binding 40-fold, and the catalytic efficiency (kcat/Km(DHF)) of the resulting protein is similar to that of Quad3. Since these Q67H mutations can mostly compensate for the K32M lesion, K32 must not be necessary for DHF binding. Another multimutant combines the K32M mutation in gene copies 1 and 3 with the Q67H mutation in all gene copies. This mutant is inhibited by DHF but not NADPH, indicating that NADPH binds only to the wild type half of the pore, while DHF can bind to either the wild type or mutant half of the pore. This inhibition pattern contrasts with the mutant containing only the Q67H substitution in all four gene copies, which is severely inhibited by both NADPH and substrate. Since gene duplication and divergence are evolutionary tools for gaining function, these constructs are a first step toward building preferences for NADPH and DHF in each half of the active site pore of this primitive enzyme.     

Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis

We have applied site-directed mutagenesis methods to change the conserved tryptophan-22 in the substrate binding site of Escherichia coli dihydrofolate reductase to phenylalanine (W22F) and histidine (W22H). The crystal structure of the W22F mutant in a binary complex with the inhibitor methotrexate has been refined at 1.9-A resolution. The W22F difference Fourier map and least-squares refinement show that structural effects of the mutation are confined to the immediate vicinity of position 22 and include an unanticipated 0.4-A movement of the methionine-20 side chain. A conserved bound water-403, suspected to play a role in the protonation of substrate DHF, has not been displaced by the mutation despite the loss of a hydrogen bond with tryptophan-22. Steady-state kinetics, stopped-flow kinetics, and primary isotope effects indicate that both mutations increase the rate of product tetrahydrofolate release, the rate-limiting step in the case of the wild-type enzyme, while slowing the rate of hydride transfer to the point where it now becomes at least partially rate determining. Steady-state kinetics show that below pH 6.8, kcat is elevated by up to 5-fold in the W22F mutant as compared with the wild-type enzyme, although kcat/Km(dihydrofolate) is lower throughout the observed pH range. For the W22H mutant, both kcat and kcat/Km(dihydrofolate) are substantially lower than the corresponding wild-type values. While both mutations weaken dihydrofolate binding, cofactor NADPH binding is not significantly altered. Fitting of the kinetic pH profiles to a general protonation scheme suggests that the proton affinity of dihydrofolate may be enhanced upon binding to the enzyme. We suggest that the function of tryptophan-22 may be to properly position the side chain of methionine-20 with respect to N5 of the substrate dihydrofolate.     

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 second-site mutation at phenylalanine-137 that increases catalytic efficiency in the mutant aspartate-27----serine Escherichia coli dihydrofolate reductase

The adaptability of Escherichia coli dihydrofolate reductase (DHFR) is being explored by identifying second-site mutations that can partially suppress the deleterious effect associated with removal of the active-site proton donor aspartic acid-27. The Asp27----serine mutant DHFR (D27S) was previously characterized and the catalytic activity found to be greatly decreased at pH 7.0 [Howell et al. (1986) Science 231, 1123-1128]. Using resistance to trimethoprim (a DHFR inhibitor) in a genetic selection procedure, we have isolated a double-mutant DHFR gene containing Asp27----Ser and Phe137----Ser mutations (D27S+F137S). The presence of the F137S mutation increases kcat approximately 3-fold and decreases Km(DHF) approximately 2-fold over D27S DHFR values. The overall effect on kcat/Km(DHF) is a 7-fold increase. The D27S+F137S double-mutant DHFR is still 500-fold less active than wild-type DHFR at pH 7. Surprisingly, Phe137 is approximately 15 A from residue 27 in the active site and is part of a beta-bulge. We propose the F137S mutation likely causes its catalytic effect by slightly altering the conformation of D27S DHFR. This supposition is supported by the observation that the F137S mutation does not have the same kinetic effect when introduced into the wild-type and D27S DHFRs, by the altered distribution of two conformers of free enzyme [see Dunn et al. (1990)] and by a preliminary difference Fourier map comparing the D27S and D27S+F137S DHFR crystal structures.     

Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase.

The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation. Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation. The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR. Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization. The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process. Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond. The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.     

Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition

Previous studies have shown that human dihydrofolate reductase (DHFR) acts as an RNA-binding protein, in which it binds to its own mRNA and, in so doing, results in translational repression. In this study, we used RNA gel mobility shift and nitrocellulose filter-binding assays to further investigate the specificity of the interaction between human DHFR protein and human DHFR mRNA. Site-directed mutagenesis was used to identify the critical amino acid residues on DHFR protein required for RNA recognition. Human His-Tag DHFR protein specifically binds to human DHFR mRNA, while unrelated proteins including thymidylate synthase, p53 and glutathione-S-transferase were unable to form a ribonucleoprotein complex with DHFR mRNA. The Cys6 residue is essential for RNA recognition, as mutation at this amino acid with either an alanine (C6A) or serine (C6S) residue almost completely abrogated RNA-binding activity. Neither one of the cysteine mutant proteins was able to repress the in vitro translation of human DHFR mRNA. Mutations at amino acids Ile7, Arg28 and Phe34, significantly reduced RNA-binding activity. An RNA footprinting analysis identified three different RNA sequences, bound to DHFR protein, ranging in size from 16 to 45 nt, while a UV cross-linking analysis isolated an ~16 nt RNA sequence bound to DHFR. These studies begin to identify the critical amino acid residues on human DHFR that mediate RNA binding either through forming direct contact points with RNA or through maintaining the protein in an optimal structure that allows for the critical RNA-binding domain to be accessible.