©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Methotrexate-resistant Variants of Human Dihydrofolate Reductase with Substitutions of Leucine 22
KINETICS, CRYSTALLOGRAPHY, AND POTENTIAL AS SELECTABLE MARKERS (*)

(Received for publication, September 27, 1994; and in revised form, December 5, 1994)

William S. Lewis (1) Vivian Cody (2) Nikolai Galitsky (2) Joseph R. Luft (2) Walter Pangborn (2) Srinivas K. Chunduru (1)(§) H. Trent Spencer (1) James R. Appleman (3) Raymond L. Blakley (1)(¶)

From the  (1)Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, the (2)Hauptman-Woodward Medical Research Institute Inc., Buffalo, New York 14203, and (3)Gensia Pharmaceuticals Inc., San Diego, California 92121

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Although substitution of tyrosine, phenylalanine, tryptophan, or arginine for leucine 22 in human dihydrofolate reductase greatly slows hydride transfer, there is little loss in overall activity (k) at pH 7.65 (except for the arginine 22 variant), but K for dihydrofolate and NADPH are increased significantly. The greatest effect, decreased binding of methotrexate to the enzyme-NADPH complex by 740- to 28,000-fold due to a large increase in the rate of methotrexate dissociation, makes these variants suitable to act as selectable markers. Affinities for four other inhibitors are also greatly decreased. Binding of methotrexate to apoenzyme is decreased much less (decreases as much as 120-fold), binding of tetrahydrofolate is decreased as much as 23-fold, and binding of dihydrofolate is decreased little or increased. Crystal structures of ternary complexes of three of the variants show that the mutations cause little perturbation of the protein backbone, of side chains of other active site residues, or of bound inhibitor. The largest structural deviations occur in the ternary complex of the arginine variant at residues 21-27 and in the orientation of the methotrexate. Tyrosine 22 and arginine 22 relieve short contacts to methotrexate and NADPH by occupying low probability conformations, but this is unnecessary for phenylalanine 22 in the piritrexim complex.


INTRODUCTION

Many variants of mammalian dihydrofolate reductase (EC 1.5.1.3, DHFR^1) are known that have a low affinity for the inhibitor methotrexate (MTX). Vectors bearing cDNA for such variants have been used to confer resistance to MTX on cells in culture (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) and in intact animals(4, 11, 12, 13, 14) . When cells are transformed or transfected with vectors carrying the appropriate cDNA, the resulting MTX resistance can be used to select transformed cells from the total population by exposure to MTX(6, 8, 9, 10, 15) .

In much of this work, cDNA for L22R mouse DHFR has been used to confer MTX resistance on cells(1, 3, 4, 9, 12, 13, 14, 15) , but this variant has low catalytic efficiency(16) . Other variant DHFRs that have been examined as such selectable markers include F31W mouse DHFR(5) , L22F hamster DHFR(6) , and F31S and F34S hDHFR(10) , and other variants of mouse DHFR(17) . However, detailed kinetic results have not been published for any of the mouse or hamster DHFR variants, and F34S hDHFR has low catalytic efficiency(18) . The fact that they provide only modest protection until amplification has generated many copies of the cDNA per cell(6, 8, 10, 19, 20, 21, 22) suggests that they are not ideal as selectable markers.

An ideal variant DHFR for conferring protection on transfected cells must not only have a very low affinity for MTX compared with the wt enzyme, but must also provide sufficient tetrahydrofolate for the needs of the cell. That is, at the usual intracellular level of DHFR, it should provide catalytic activity comparable with that of wt DHFR under normal conditions. This would obviate the need for amplification of the mutant cDNA after its integration into the cell genome. Further, the variant protein must be sufficiently stable that the concentration of the active form in the cell can be maintained without an increased rate of expression.

Since previous studies (17, 23, 24) have indicated that variants of mammalian DHFR with substitution of bulky amino acids for Leu may be good selectable markers, we present here detailed analysis of four such variants of hDHFR that we have produced by site-directed mutagenesis and expression in Escherichia coli. All of these variants exhibit good stability and markedly decreased affinity for MTX. Moreover, only the Arg variant suffers from a large decrease in catalytic efficiency at physiological pH. X-ray crystal structures have revealed the molecular changes that are present in three of the variants.


EXPERIMENTAL PROCEDURES

Materials

Unless otherwise noted, material used were as described previously(25, 26, 27) . Methotrexate and aminopterin were from Sigma. 10-Deaza-10-ethylaminopterin, trimetrexate, and piritrexim were gifts from Ciba-Geigy, Warner Lambert-Parke Davis, and BurroughsWellcome, respectively. The pDS5/hDHFR plasmid was a gift from Dr. D. Stüber of Hoffmann La Roche.

Construction and Expression of Mutants of hDHFR

Mutations were introduced into the cDNA and verified by sequencing as described previously(28, 29) . After expression in M15(pREP4) E. coli, the enzyme was purified as described previously(29) .

Purity and Concentration of Enzyme

Polyacrylamide gel electrophoresis of each of the purified variant enzymes showed a single band after staining with Coomassie Blue. The concentration of the binding sites of the purified enzyme was determined by MTX titration of protein fluorescence at 330 nm (excitation at 280) with monitoring on an SLM Aminco 8000 spectrofluorimeter.

Steady State Kinetics

Unless otherwise indicated, hDHFR activity was assayed in the presence of 100 µM dihydrofolate, 100 µM NADPH in MATS buffer (25 mM MES, 25 mM acetate, 50 mM Tris, 100 mM sodium chloride, and 0.02% sodium azide) pH 7.65, at 20 °C as described (29) using a Beckman DU-7500 spectrophotometer.

In the case of variants with low K(m) values for dihydrofolate, the steady state parameters k and K(m) were determined by the use of an Applied Photophysics stopped-flow spectrophotometer. Solutions in both syringes contained 0.29 M 2-mercaptoethanol in MATS buffer, pH 7.65 at 20 °C. The resulting velocities were fit to the Michaelis-Menten equation using the Kaleidagraph program (Abelbeck Software). K(m) for NADPH was determined similarly. For the L22F variant, the dependence of activity on the concentration of the substrates was determined in the same way, but data were fit to the equation for substrate inhibition(18) .

Determination of Enzyme Stability

The enzyme was incubated at 37 °C in a solution consisting of 250 nM enzyme, 1 mM NADPH, 100 mM potassium phosphate, pH 7.4. Samples were removed at intervals and assayed for enzymic activity under standard conditions.

K(i) for Inhibitors

K(i) for various inhibitors was determined as described previously(30) . K(i) is obtained from the steady state activity at various inhibitor concentrations and since K(i)/K(i) = 1 + [dihydrofolate]/K(m), K(m) for dihydrofolate must be known. In the case of the L22F variant, an ``operational'' K(m) was approximated by fitting activity at various dihydrofolate concentrations up to 10 µM to the Michaelis-Menten equation, without taking account of the substrate inhibition occurring. It should be noted that 10 µM is the concentration of dihydrofolate at which K(i) values were measured and is also the concentration at which activity is a maximum.

Equilibrium Dissociation Constants

The equilibrium dissociation constants (K(d)) for the binary complexes of dihydrofolate and MTX were determined by titration of protein fluorescence at 330 nm with ligand in MATS buffer, pH 7.65 at 20 °C (30) . Excitation was at 280 nm. Determination of K(d) for binding of MTX to the enzyme-NADP complex was performed similarly. For determination of K(d) for the binary complex of tetrahydrofolate, 0.29 M 2-mercaptoethanol was included in the solution.

K(d) for the binary complex with NADPH was determined from the transfer energy fluorescence at 435 nm(31) . K(d) for binding of MTX to the enzyme-NADPH complex was determined by measuring MTX quenching of this transfer energy.

Determination of the Rate Constant for Chemical Transformation

The chemical transformation rate constant (k) was determined by a modification of the method used previously(32) . Final concentrations were: 2.5 µM enzyme, 100 µM NADPH, 0.29 M 2-mercaptoethanol, and 0.2-1.5 µM dihydrofolate in MATS buffer at pH 7.65. In a parallel series of experiments, final concentrations were: 0.2-1.5 µM NADPH, 100 µM dihydrofolate, and 2.5 µM enzyme. Independent values of k were obtained in the two series of experiments. The data were fit to a second order binding reaction plus the chemical transformation step by a modification of the CRICF program(33) .

Computer simulations of the reaction course were performed as described previously(25) , with the use of k, k, and k values obtained from the data analysis and experimental concentrations. A few data sets that did not agree well with simulation were discarded, and the data fitting routine was repeated without them.

In some experiments, NADPD replaced NADPH in the appropriate syringe. The isotope effect on k (^Dk) was obtained by calculating the ratio of k in the presence of NADPH to k in the presence of NADPD.

Determination of Methotrexate Binding Rates

The rate constants for MTX binding to the enzymebulletNADPH complex (k) and release from the enzymebulletNADPHbulletMTX complex (k) were determined by stopped-flow fluorometric methods described previously(30, 33) .

In many cases, the thermodynamic equilibrium constant, k/k, is much higher than the corresponding values of K(i). This can be accounted for by assuming isomerization of the initial ternary complex to a nondissociating conformer(31) , according to the equation:

where the asterisk indicates the nondissociating conformer. The thermodynamic equilibrium constant for this isomerization step, K (k/k(r)), can be calculated by the equation: K = (k/k K(i)) - 1.

The apparent dissociation rate constant for release of MTX (k) from both isomers of the DHFRbulletNADPHbulletMTX complex was determined by competition with 10-deaza-10-ethylaminopterin as described previously(26, 30) .

Crystallization and Structure Analysis Methods

Crystals of each variant complex were grown with a molar excess of NADPH and inhibitor together with the enzyme in 0.1 M phosphate buffer, pH 8.0, and 62% ammonium sulfate. Hanging drop experiments were carried out at room temperature. All crystals grew in a rhombohedral system, space group R3 with hexagonal indexing, isomorphous with the wt hDHFR binary (34) and ternary (35) complexes. L22Y hDHFRbulletMTXbulletNADPH: a = 87.496, c = 76.733 Å; resolution, 1.90 Å; R = 19.1%, 12027 reflections from 8.0-1.90 Å. L22F hDHFRbulletPTXbulletNADPH: a = 87.002, c = 76.728 Å; resolution 2.0 Å; R = 16.4%, 10046 reflections from 8.0-2.0 Å. L22R hDHFRbulletMTXbulletNADPH: a = 86.058, c = 77.677 Å; resolution, 2.2 Å; current refinement, R = 21.0%, 6383 reflections from 8.0-2.3 Å. Data were collected on a Raxis IIc imaging plate system.

Since the crystals were isomorphous with those of wt hDHFR ternary complex with MTXT(35) , hDHFR coordinates from this structure were used to calculate phases for the structure determination. Refinement was continued using the restrained least square program PROLSQ (36, 37) in combination with the model building program CHAIN(38) . Current refinement for these structures is nearly complete to the resolution of the data, and the detailed analysis of these structures will be reported elsewhere.


RESULTS

Expression and Purification of Variants

DHFR constituted about 7% of the soluble protein in the extracts of E. coli. The purified enzymes were homogeneous as judged by gel electrophoresis following staining with Coomassie Blue. The concentration of the binding sites as determined by titration with MTX is in good agreement (>90%) with the concentration of the enzyme determined both by UV absorbance at 280 nm (27) and colorimetric protein assay(39) . Tightly bound folate and degradation products were completely removed by preparative isoelectric focusing as judged by a ratio of absorbance at 280 nm to that at 320 nm > 20. All of the variants have reasonable stability under the conditions used. L22Y and L22F have half-lives comparable to that of wt hDHFR. L22W is slightly less stable than the wt enzyme, while L22R has a half-life 2.6 times that of wt hDHFR.

Steady State Kinetics

Values of K(m) for NADPH and dihydrofolate and k are given in Table 1. Initial velocity for the L22F variant decreased as the dihydrofolate concentration was increased above 10 µM. Substrate inhibition has been observed with other hDHFR variants(18) , the data were fit to the equation used previously, and the two values of k and K(m) were calculated as described previously(18) . By analogy with other variants which exhibit substrate inhibition(18) , at low dihydrofolate concentrations, the predominant pathway is probably through EbulletNADPHbulletH(2)folate EbulletNADPbulletH(4)folate EbulletNADP E EbulletNADPH, whereas at high dihydrofolate concentrations the pathway going through EbulletNADPHbulletH(2)folate EbulletNADPbulletH(4)folate EbulletNADP EbulletNADPbulletH(2)folate EbulletH(2)folate predominates. k values for the other variants are all lower than the value for wt hDHFR, and almost zero for the L22R variant at pH 7.65. K(m) for both substrates is increased for all of the variants, but the range of K(m) values is smaller than that for k values. Unlike wt hDHFR, none of these variants exhibit transient state hysteresis since k is the major rate-limiting step and is common to both pathways.



Effect of pH on Activity

Despite its low activity at pH 7.65, the L22R variant has activity comparable to wt and the other variants at low pH (Fig. 1), and, in fact, all of the variants display greater pH dependence of activity than does wt. Data for the L22R and L22W variants fit well to an equation containing a single pK (4.9 and 6.0, respectively). The L22F and L22Y variants require an equation with two pK values: 4.8 and 7.1 for L22Y and 5.2 and 7.7 for L22F. The pK values obtained from these data probably do not correspond to true ionization constants, but to the pH at which the rates of two rate-limiting steps become equal(40) .


Figure 1: Dependence of activity on pH. Initial rates observed in 100 µM dihydrofolate, 100 µM NADPH in MATS buffer at the indicated pH at 20 °C. For L22W and L22R, lines show the fit of data to: activity = a/(1 + 10) and for L22Y and L22F lines show the fit of data to: activity = a/(1 + 10) + b/(1 + 10). The broken line shows the pH dependence for wt hDHFR.



Rate Constant for the Chemical Transformation

The rate constant for the chemical transformation step, k, is also given in Table 1. For all four variants, k is greatly decreased compared with wt, the decrease being much more than for k.

In the case of the L22W and L22R variants, the close agreement between k and k indicate that the chemical transformation step is rate-limiting at steady state, and this is confirmed by the high values of ^Dk which are not significantly lower than those for ^Dk. For L22Y, k is lower than k, and ^Dk < ^Dk, so that the steady state rate is limited partly by the chemical transformation step and partly by some subsequent step, presumably product release. However, for the L22F variant, ^Dk is quite similar to ^Dk so that the chemical transformation is rate-limiting, and a good approximation for k, the overall rate constant for the faster pathway, is given by k. This permits an approximation of K(m) for dihydrofolate(18) . For the L22R variant, k, like k, increases markedly as the pH is lowered, reaching 4.9 ± 0.8 s at pH 5.0. ^Dk at pH 5.0 (2.6 ± 0.2) is not significantly different from ^Dk (2.9 ± 0.3), so that the chemical transformation is rate-limiting.

Equilibrium Dissociation Constants (K(d))

The K(m) values for both substrates are greater than those of the wt enzyme (Table 2). However, since the value of K(m) depends on other rate constants besides k and k for the binding of the substrate, the equilibrium dissociation constants, K(d), for binary substrate or product complexes were determined (Table 2). The binary K(d) for dihydrofolate is lower than the wt value for three of the variants, but is more than doubled for the L22F variant. In contrast to the increased affinity for dihydrofolate, tetrahydrofolate is bound less tightly by all the variant hDHFRs, with K(d) increasing 5- to 16-fold. NADPH and NADP are bound somewhat less tightly by the variant DHFRs than by wt.



Affinity of the unliganded variant enzymes for MTX is only slightly lower than that of wt (Table 2), except in the case of L22R where K(d) is 120-fold higher than K(d) for wt. However, the affinity of MTX for NADP complexes of all the variant hDHFRs is much lower than for the NADP complex of wt hDHFR (by factors of 20 to 300), and the affinity of MTX for the ternary NADPH complexes is still less (K(d) 740- to 28,000-fold higher than for wt).

Inhibition Constants (K(i))

In all cases, the affinity of the variant hDHFRs for the inhibitors, as measured by K(i), is decreased in comparison with wt hDHFR, the extent of the decrease varying considerably (factors of 33 to 960,000) with the variant and the inhibitor (Table 3). The decrease in the affinity of L22Y for aminopterin is less (K(i) increased 240-fold above wt K(i)) than for the other inhibitors, for which decreases are similar. L22F also has the smallest decrease in affinity for aminopterin, with affinity for trimetrexate and piritrexim decreasing less than for MTX and 10-deaza-10-ethylaminopterin. Substrate inhibition complicates determination of K(i) for L22F, since K(i) is dependent on K(m). At the concentration of dihydrofolate present during the determination of K(i), both pathways would be operational in an unknown ratio. An operational K(m), corresponding to the combined activity of the two pathways, determined as described under ``Experimental Procedures,'' was therefore used. The rather small discrepancy between K(i) and K(d) or k/k for MTX binding to L22F (0.546, 1.86, and 0.602 nM, respectively) suggests that the error in K(m) is not very large.



The magnitude of the affinity decreases for L22W for inhibitors are intermediate between those for L22Y and L22F, with the notable exception of the affinity of L22W for piritrexim, which is decreased by only a small factor. The affinity of L22R for MTX is decreased by a factor comparable to that for the other variants, but the affinity of L22R for the other inhibitors is decreased more than for any other variant.

Rate Constants for MTX Binding to and Release from Variant hDHFRs

Values of k and k, determined in binding experiments with enzymebulletNADPH, are shown in Table 4. k values for the formation of complexes with MTX fall within a fairly narrow range and are only slightly less than for the wt complex. Values of k obtained by this method for MTX dissociation from its ternary complexes are comparable for the four variants and about 2500 times that for wt enzyme (Table 4). Consequently, the dissociation constants obtained by this method for the initial ternary complex are also quite similar for the four variants, but much higher than that for wt.



Two pieces of evidence indicate that formation of the ternary complex cannot be a simple binding reaction. First, k/k K(i); second, k k, the rate constant for the release of MTX from the ternary complex obtained in competition experiments (Table 4). Binding appears to be followed by isomerization of the initial ternary complex to a nondissociating conformer ()(33) . Values of K, the equilibrium constant for the isomerization, calculated as indicated under ``Experimental Procedures,'' are shown in Table 4.

In the direct measurement of MTX binding to enzymes by stopped-flow fluorimetry, only one reaction phase, corresponding to the binding step, was observed. An expected, second phase corresponding to isomerization of the initial complex, and consequent additional ligand binding, was not observed even under conditions where it was calculated from the constants in Table 4that additional binding due to the isomerization accounted for a large fraction of the total binding. However, this second phase would only be observed under the experimental conditions if k < k[MTX]. This condition is likely to occur only at high MTX concentration, where the amplitude of the second phase would be very small.

Crystal Structures

Crystallization of three variants (L22Y, L22F, L22R) was carried out in the presence of various inhibitors and NADPH. The three complexes for which structures are reported here are the first ones for each variant for which well-diffracting crystals were obtained. All three crystals are ternary complexes with NADPH also present.

The crystal structures for the complexes are compared with the wt hDHFRbulletMTTbulletNADPH complex(35) , by least squares superposition of all backbone atoms (Profit program, G. D. Smith, Hauptman-Woodward Medical Research Institute Library). The root mean square deviation in backbone position in the variant complex from those of the wt complex with which it was paired, were as follows: L22Y hDHFRbulletMTXbulletNADPH, 0.15 Å; L22F hDHFRbulletPTXbulletNADPH, 0.20 Å; L22R hDHFRbullet MTXbulletNADPH, 0.25 Å. As these values indicate, there is little perturbation of the protein structure apart from the known change in residue 22.

The active site regions of the three complexes are shown in Fig. 2Fig. 3Fig. 4. As shown in Fig. 2, the largest differences between L22R hDHFRbulletMTXbulletNADPH and the wt ternary complex is observed for the backbone and side chain positions of residues 21-27, which have an average backbone deviation of 0.89 Å. This is the largest difference in individual backbone positions when the variants are compared with the wt. There is little perturbation of the backbone or side chain structure in the vicinity of the active site of variants L22F and L22Y ( Fig. 3and Fig. 4). However, the unusual binding geometry for PTX in the complex with the L22F variant results in a large shift in Phe to relieve the steric constraints of interactions with the methoxy substituents. Similar changes were noted in the ternary complex of avian DHFR and trimethoprim(41) . The largest shifts in the backbone of the L22F variant are near residue 63 in a flexible loop region not shown in Fig. 3. The largest shifts in the backbone in L22Y are near residue 44, also in a flexible loop region not shown in Fig. 4.


Figure 2: Crystal structure of L22R hDHFR. Stereoscopic view of the active site region of the crystal structure of L22R hDHFRbulletMTXbulletNADPH (solid lines). The corresponding region of the crystal structure of wt hDHFRbulletMTXTbulletNADPH (dotted lines) (35) is shown for comparison.




Figure 3: Crystal structure of L22F hDHFR. Stereoscopic view of the active site region of the crystal structure of L22F hDHFRbulletPTXbulletNADPH (solid lines). The corresponding region of the crystal structure of wt hDHFRbulletMTXTbulletNADPH (dotted lines) (35) is shown for comparison.




Figure 4: Crystal structure of L22Y hDHFR. Stereoscopic view of the active site region of the crystal structure of L22Y hDHFRbulletMTXbulletNADPH (solid lines). The corresponding region of the crystal structure of wt hDHFRbulletMTXTbulletNADPH (dotted lines) (35) is shown for comparison.



As noted (Table 5), in the structures of L22Y hDHFRbullet MTXbulletNADPH and L22R hDHFRbulletMTXbulletNADPH, the side chains at position 22 adopt low probability conformations in order to avoid unfavorable intermolecular contacts with the inhibitor and cofactor. In the case of L22F hDHFRbullet PTXbulletNADPH, the change in binding orientation of PTX compared with that of MTX permits Phe to adapt a high probability conformation in the active site.




DISCUSSION

Effect of Mutations on the Chemical Transformation and Catalytic Activity

Although many variants of hDHFR have very large decreases in k, k is often little affected (as L22F, L22Y, and L22W) because product dissociation is much slower than the chemical transformation for wt hDHFR. In the case of L22R, the decrease in k at pH 7.65 is so great (k 3 times 10 that of wt) that k is also very low (4 times 10 that of wt).

Since there appears to be a full isotope effect on k for all these variants, the rate constant for hydride transfer is decreased. One possible mechanism for these effects on k is a change in distance between the hydride donor and acceptor atoms, i.e. C4 of the nicotinamide ring of bound NADPH and C6 of bound dihydrofolate, or C7 of bound folate. It has been calculated that an increase in the C-C distance from its optimum of 2.6 Å by 0.1 or 0.3 Å increases the activation energy for hydride transfer by 0.7 and 5 kcal/mol(42) , respectively.

pH Dependence of k for Variant DHFRs

The variants all show far greater changes in activity with changing pH than the wt enzyme does (Fig. 1). Since the measurement of activity was made at high concentrations of dihydrofolate and NADPH, the pH effects are unlikely to be due to changes in substrate binding. Instead, the effects are most likely due to pH-induced changes in the rate of hydride transfer which increases as the pH is lowered(^2), in H(4)folate release which decreases as the pH is lowered(43) , or in NADP release, or in combinations of these. If an increase in k with decreased pH is the major effect, then it appears that the pK(a) governing this effect of pH is considerably lower than for wt hDHFR, for which it is 6.2.^2 This in turn suggests that the pK(a) for N5 of dihydrofolate bound in the ternary substrate complex of the variants is significantly lower than its pK(a) of 6.3 in the wt hDHFR ternary complex (44) . This changed pK(a) in part accounts for the lowered k at pH 7.65 for the variant hDHFR.

The additional apparent pK(a) seen for L22Y and L22F is perhaps due to the effect of pH on H(4)folate dissociation, but the high activity of all four variants at pH < 5, compared with wt, suggests that k for H(4)folate is higher for the variants than for wt. This is consistent with the 5- to 23-fold higher K(d) values for H(4)folate binary complexes of the variant hDHFRs than for the wt complex (Table 2).

Effects of Mutations on Binding of Substrates and Products

As in the case of mutations causing substitution of smaller residues for Phe(29) or Phe(18) , the mutations replacing Leu did not result in as great a decrease in affinity for dihydrofolate as for H(4)folate (Table 2). In fact, as in the case of substitutions for Phe, the affinity of the apoenzyme for dihydrofolate was actually greater than that of wt in the case of L22Y, L22W, and L22R. Whether the greater sensitivity of H(4)folate binding to changes in the binding pocket is due to the puckered reduced pteridine ring of H(4)folate, to the space occupied by its additional hydrogens, or to some other mechanism, remains unclear. However, the result is that K(m) for dihydrofolate is not increased as much as would otherwise be the case.

There is a moderate decrease in the affinity of the L22Y apoenzyme for NADPH and NADP (Table 2). This may be related to loss of the binding interactions of CD1 of Leu with C5 of the nicotinamide ring of NADPH in the wt hDHFRbulletNADPHbulletMTXT complex. In the corresponding complex of the L22Y variant, little interaction occurs between Tyr and the nicotinamide ring, the shortest interatomic distance being 4.22 Å. The large (40-fold) increase in K(d) for the NADPH complex of the L22W variant may perhaps reflect steric interference with binding of the nicotinamide ring of NADPH by the tryptophan side chain.

The mutation-dependent increases in binary K(d) values for H(4)folate are larger than those for NADP (Table 2). If the mutations similarly affect dissociation of the ternary product complexes, EbulletNADPbulletH(4)folate, then the preferred mechanistic pathway will probably be through EbulletNADP, with H(4)folate dissociating first, as in the case of mutants with substitutions for Phe(18) . Substrate inhibition will occur if k for dihydrofolate association with EbulletNADP is greater than k for NADP dissociation from this complex and if the subsequent dissociation of dihydrofolate is rate-limiting. This is the case for L22F hDHFR, where dihydrofolate inhibition occurs, but not for the other variants. The higher K(d) values for the EbulletNADP complexes of the L22W and L22R variants suggests that a higher k for NADP is at least partly responsible for the absence of substrate inhibition for these variants.

Effect of Mutations on Inhibitor Binding

As in the case of other variants that we have examined in some detail(18, 29) , variants with substitutions for Leu exhibit much greater decreases in affinity for inhibitors than for dihydrofolate or H(4)folate ( Table 2and Table 3). Large decreases in affinity occur not only for MTX but for four other inhibitors that are of clinical interest (Table 3).

The decrease in the affinity of MTX for the variant apoenzymes is quite small compared with that for wt apoenzyme: it is only the affinity of MTX for the enzymebulletNADPH complex that is greatly decreased by the mutations. In the case of wt hDHFR, the affinity of MTX is 480-fold greater for the enzymebulletNADPH complex than for the apoenzyme, but in the case of the variant enzymes, complex formation with NADPH has little effect on MTX affinity (Table 2). These results are in contrast to those for F31G hDHFR(29) . The affinity of MTX for the apoenzyme of the latter is decreased 37-fold compared with wt, but because of greater binding to the EbulletNADPH complex, MTX affinity for the latter is only decreased 130-fold compared with wt. For the F34A variant, the contrast with the present results is even greater, since K(d) for the binary MTX complex is increased 8,000-fold compared with wt, and for the ternary complex 18,000-fold(18) .

The lack of cooperativity in the ternary MTX complexes of the variants with substitutions for Leu may perhaps be due in part to slightly greater separation between the nicotinamide and pteridine rings. The distance of C4 of the nicotinamide ring from C6 of the pteridine ring is 3.93 and 4.27 Å, respectively, for wt and L22Y hDHFR. Similarly, the C4-C7 distances are 4.22 and 4.52 Å, respectively. (For numbering of MTX atoms, see (45) .)

The decrease in affinity of MTX for hDHFR in the ternary complex can be due to a decrease in k, an increase in k, or a decrease in K (). For all the variant hDHFRs, k is decreased a little, k is greatly increased (2,300 to 4,100-fold) with the result that k/k is greatly increased in all cases. For all the variants, the occurrence of isomerization strengthens binding, especially in the case of L22Y and L22W, and only in the case of L22R is K less than for wt.

Relation of Changes in MTX Binding to Structure

It is of interest to examine the crystallographic structures with a view to providing molecular interpretations of the decreased affinity of MTX for the variant hDHFRs. The most valid comparisons are of the wt hDHFRbulletNADPHbulletMTXT structure with that of L22Y hDHFRbulletNADPHbulletMTX, and with that of L22R hDHFRbulletNADPHbulletMTX. Decreased affinity is due primarily to an increase in k, and this might be due to decreased affinity between MTX and active site side chains, or to increased diffusion of MTX from the active site.

If the interactions with MTX of the residues at position 22 are first compared, there seems to be little difference in the number of close contacts in the L22Y and wt complexes, but there is virtually no interaction between the Arg side chain and bound MTX. In the wt complex, the closest interactions are between C7, C6, and N8 of MTX with CD1, CD2, and CD2 of Leu, respectively, with separations of 4.41, 3.60, and 4.07 Å, respectively. In the case of the L22Y complex, the closest contacts are between the 10-methyl group of MTX and CD2 and CE2 of Tyr, and between C7 of MTX and CD2 of Tyr, with separations of 4.27, 4.58, and 3.99 Å, respectively. By contrast, the closest approach of atoms of the Arg side chain and MTX is for CG of Arg and the 10-methyl of MTX (5.37 Å). Thus binding energy is lost at this residue for the L22R variant and explains some of the loss of affinity of MTX for this variant, but not for the L22Y variant. There is no clear evidence for decreased interaction of side chains of other residues, such as Phe and Phe.

The molecular basis of the greatly increased k for MTX dissociation from complexes of the variants might also be sought in structural factors that facilitate the diffusion of the inhibitor out of the binding site. McTigue et al.(45) have suggested that diffusion of a pteridine out of the binding site requires the phenyl side chain of Phe to rotate about the Calpha-Cbeta bond from the position in the ternary complex, so that the pteridine ring can exit between Phe and Leu. This rotation of Phe should be unhindered in the variants, and indeed in the crystal structure of the L22F complex (Fig. 3) the Phe side chain is seen in its alternative position. It should be noted, however, that the Phe and Tyr side chains are considerably closer to Phe than is Leu. The activation energy for MTX dissociation may also be lowered somewhat by the adoption of lower energy conformations by Tyr and Arg when the pteridine binding site is emptied.

It seems probable that contributions from these and other small structural effects of the mutations together produce the overall increase in k.

Suitability of Variants for Conferring Resistance to MTX on Cells

The variants with substitutions for Leu appear eminently suitable for making cells MTX-resistant according to many criteria: they have good stability in vitro; three of them have inhibition constants (K(i)) considerably higher than those of the F31S and F31G variants of hDHFR (K(i) 0.24 and 0.35 nM, respectively), both of which have been shown to confer significant protection to cells in culture(10, 11, 46) ; and they have catalytic efficiencies (k/K(m)) that compare favorably with that of F34S hDHFR (18) which has also been reported to confer resistance on cells(10) . k/K(m) values for L22Y, L22F, L22W, and L22R are 12, 15, 10, and 0.03 s µM, respectively, and for F34S is 0.017 s µM. Our initial results with the L22Y variant (46) indicate that transfection of the cDNA into mouse fibroblasts on a retroviral vector does indeed afford considerable protection from MTX.


FOOTNOTES

*
This research was supported in part by United States Public Health Service Research Grant R01 CA 31922 (to R. L. B. and V. C.), Cancer Core Grant P30 CA 21765 (to R. L. B.), by a St. Jude Children's Research Hospital Special Fellowship (to W. S. L.), and by the American Lebanese Syrian Associated Charities (ALSAC) (to R. L. B. and W. S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

§
Present address: Dept. of Microbiology, University of Pennsylvania, 209 Johnson Pavilion, Philadelphia, PA 19104-6076.

To whom correspondence and reprint requests should be addressed: Dept. of Molecular Pharmacology, St. Jude Children's Research Hospital, P.O. Box 318, Memphis, TN 38101. Tel.: 901-522-0359; Fax: 901-531-2394.

(^1)
The abbreviations used are: DHFR, dihydrofolate reductase; hDHFR, human DHFR; wt, wild type DHFR; H(2)folate, 7,8-dihydrofolate; H(4)folate, (6S)-5,6,7,8-tetrahydrofolate; aminopterin, 4-amino-4-deoxyfolic acid; MTX, methotrexate, 4-amino-4-deoxy-10-methylfolic acid; MTXT, 4-amino-4-deoxypteroyl--(1H-tetrazol-5-yl)-L-alpha-aminobutyric acid; PTX, piritrexim, 2,4-diamino-6-(2,5-dimethoxybenzyl-5-methylpyrido[2,3-d]pyrimidine; trimetrexate, 2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline; MES, 2-(N-morpholino) ethanesulfonic acid; NADPD, (4R)-[^2H]NADPH; L22X, variant DHFR in which the leucine at position 22 has been replaced with the amino acid X; k, rate constant for chemical transformation step.

(^2)
J. R. Appleman and R. L. Blakley, unpublished results.


ACKNOWLEDGEMENTS

We thank J. Clay McCastlain for producing and cloning three of the mutant cDNAs.


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