(Received for publication, September 27, 1994; and in revised form, December 5, 1994)
From the
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.
Many variants of mammalian dihydrofolate reductase (EC 1.5.1.3,
DHFR) 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.
In
the case of variants with low K values for
dihydrofolate, the steady state parameters k
and K
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
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) .
K for the binary complex with NADPH was
determined from the transfer energy fluorescence at 435
nm(31) . K
for binding of MTX to the
enzyme-NADPH complex was determined by measuring MTX quenching of this
transfer energy.
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 (
k
) was obtained by calculating the
ratio of k
in the presence of NADPH to k
in the presence of NADPD.
In many cases, the
thermodynamic equilibrium constant, k/k
, is much higher than
the corresponding values of K
. 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
),
can be calculated by the equation: K
= (k
/k
K
)
- 1.
The apparent dissociation rate constant for release of
MTX (k) from both isomers of the
DHFR
NADPH
MTX complex was determined by competition with
10-deaza-10-ethylaminopterin as described
previously(26, 30) .
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.
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.
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
k
which are not significantly lower than those for
k
. For L22Y, k
is
lower than k
, and
k
<
k
, 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,
k
is quite similar to
k
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
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.
k
at pH 5.0
(2.6 ± 0.2) is not significantly different from
k
(2.9 ± 0.3), so that the chemical
transformation is rate-limiting.
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 is 120-fold higher than K
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
740- to 28,000-fold higher than for wt).
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.
Two pieces of evidence
indicate that formation of the ternary complex cannot be a simple
binding reaction. First, k/k
K
; 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.
The crystal structures for the
complexes are compared with the wt hDHFRMTT
NADPH
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 hDHFR
MTX
NADPH, 0.15 Å;
L22F hDHFR
PTX
NADPH, 0.20 Å; L22R hDHFR
MTX
NADPH, 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 hDHFRMTX
NADPH 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 hDHFRMTX
NADPH (solid lines). The
corresponding region of the crystal structure of wt
hDHFR
MTXT
NADPH (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 hDHFRPTX
NADPH (solid lines). The
corresponding region of the crystal structure of wt
hDHFR
MTXT
NADPH (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 hDHFRMTX
NADPH (solid lines). The
corresponding region of the crystal structure of wt
hDHFR
MTXT
NADPH (dotted lines) (35) is
shown for comparison.
As noted (Table 5), in
the structures of L22Y hDHFR MTX
NADPH and L22R
hDHFR
MTX
NADPH, 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
hDHFR
PTX
NADPH, the change in binding orientation of PTX
compared with that of MTX permits Phe
to adapt a high
probability conformation in the active site.
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.
The additional apparent pK seen for L22Y
and L22F is perhaps due to the effect of pH on H
folate
dissociation, but the high activity of all four variants at pH < 5,
compared with wt, suggests that k
for
H
folate is higher for the variants than for wt. This is
consistent with the 5- to 23-fold higher K
values
for H
folate binary complexes of the variant hDHFRs than for
the wt complex (Table 2).
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 hDHFR
NADPH
MTXT
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
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 values
for H
folate are larger than those for NADP (Table 2).
If the mutations similarly affect dissociation of the ternary product
complexes, E
NADP
H
folate, then the
preferred mechanistic pathway will probably be through E
NADP, with H
folate dissociating first, as
in the case of mutants with substitutions for
Phe
(18) . Substrate inhibition will occur if k
for dihydrofolate association with E
NADP 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
values for the E
NADP
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.
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 enzymeNADPH
complex that is greatly decreased by the mutations. In the case of wt
hDHFR, the affinity of MTX is 480-fold greater for the
enzyme
NADPH 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 E
NADPH 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
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.
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 C
-C
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.
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.