contact information for elizabeth e. howell: phone, 865-974-4507

Role of K32 Residues in R67 Dihydrofolate Reductase

Probed by Asymmetric Mutations†

Stephanie N. Hicks, R. Derike Smiley, Lori G. Stinnett,

Kenneth H. Minor and Elizabeth E. Howell*

‡Department of Biochemistry, Cellular and Molecular Biology

University of Tennessee, Knoxville, TN 37996-0840

Running Title: Asymmetric K32M Mutations in R67 DHFR

Keywords: tetrahydrofolate dehydrogenase, trimethoprim, site-directed mutagenesis, binding

specificity, enzyme evolution, gene quadruplication, salt bridges, ionic strength dependence, salt

effects, ionic interactions, symmetry, non-productive binding.

† This research was supported by NSF grant MCB-0131394 (to E.E.H.).

Contact Information for Elizabeth E. Howell: phone, 865-974-4507; fax, 865-974-6306, email,

[email protected].

JBC Papers in Press. Published on August 27, 2004 as Manuscript M404484200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARY

R67 dihydrofolate reductase (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 et al., (2003) Biochemistry 42,

10569-78], however the residue(s) that participates in ionic contacts with the negatively charged

tail of DHF as well as the phosphate groups in NADPH was not identified. Several studies

predict that K32 residues were involved, however mutations at this residue destabilize the R67

DHFR homotetramer. To study the role of K32 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, while 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 2 mutations on the same half pore,

enhances kcat four to five fold compared to the parent enzyme, albeit at the expense of weaker

binding of ligands. Since 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 K32 and the charged tail of dihydrofolate. This unusual catalytic

scenario arises from the 222 symmetry imposed on the single active site pore.

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INTRODUCTION

R67 dihydrofolate reductase (R67 DHFR) is an R-plasmid encoded enzyme that catalyzes

the NADPH dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). Its presence

in bacteria confers resistance to the antibiotic, trimethoprim. This enzyme is not similar in

sequence or structure to the chromosomally ended DHFRs.

R67 DHFR is a homotetramer and the pore that traverses the length of the molecule is the

active site. Surprisingly the structure possesses 222 symmetry (1), which imposes numerous

constraints on binding and catalysis. For example, the symmetry requires that for each binding

site, there must be three additional, symmetry related sites. However solution studies find only

two sites can be occupied simultaneously because of steric constraints. The possible binding

combinations are two NADPH molecules, or two folate/DHF molecules or one NADPH plus one

folate/DHF molecule (2). Only the latter is productive. Thus binding of neither ligand can be

optimized and a “one site fits both” approach is employed (3,4). Another constraint arising from

the symmetry is that addition of a mutation to the gene results in 4 mutations per single active

site pore. While addition of one mutation might help DHF binding in one half the pore, it will

not necessarily help NADPH binding in the other half of the pore (or vice versa). Breaking the

222 symmetry of R67 DHFR by introduction of asymmetric mutations should help unravel how

R67 DHFR functions.

To allow introduction of asymmetric mutations, a tandem array of four, in-frame R67

DHFR gene copies has been constructed (5,6). The resulting protein product (named Quad3)

possesses a mass four times that of the R67 DHFR monomer. Each domain in Quad3

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corresponds to a monomer in R67 DHFR. The linker sequence connecting the gene copies is the

natural N-terminus. The quadruplicated gene product is almost fully active with only a slight

decrease in kcat (1.6 fold) and a slight increase in Km values (1.5 fold). All other studies suggest

the quadruplicated gene product mimics the homotetramer very closely.

Which mutations should be introduced into Quad3 to allow analysis of R67 DHFR

catalysis? Previous studies have identified K32, Q67, I68 and Y69 as the most critical residues

in binding and catalysis2 (4,7-9). Therefore these residues were targeted for introduction of

asymmetric mutations. The first asymmetric mutation studied was Q67H (6). This study focuses

on the role of K32 residues and a third study considers the effect of Y69F mutations (Stinnett et

al., see companion manuscript).

What role is proposed for K32? Since both substrates are negatively charged (DHF

possesses two carboxylate groups in its p-aminobenzoyl–glutamic acid (pABA-glu) tail and

NADPH possesses a 2’ phosphate on the AMP ribose as well as the pyrophosphate bridge), the

role of counter charges was considered. The only positively charged residue in the active site is

K32 and the symmetry related positions of this residue are shown in Figure 1. To examine the

role of K32 in R67 DHFR function, mutagenesis was employed. Unfortunately, all mutants

destabilized the homotetramer, therefore salt effects were used to indirectly probe the role of

K32 (7,10). Increasing ionic strength was found to dramatically increase the Km values for both

ligands. A smaller, but significant rise in kcat was also noted, indicating ionic interactions are

involved in binding and catalysis (11,12).

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Further support for the importance of K32 in binding and catalysis comes from NMR

experiments which find a chemical shift in the resonances associated with K32 upon NADPH

binding (13). Also, docking of NADPH into the R67 DHFR•folate complex predicts that K32

likely forms an ionic interaction with the 2’phosphate of NADPH, while a symmetry related K32

likely interacts with the γ-carboxyl group on the glutamate tail of DHF (3). Our simple model

for binding and catalysis suggests K32 guides the negatively charged ligands to the active site

pore by establishing a positive electrostatic potential (3). Once the ligands near the active site,

symmetry related K32 residues form direct ionic interactions with both NADPH and DHF. After

formation of the ground state complex, at least one ionic interaction breaks, leading to hydride

transfer.

To evaluate the role of K32 in R67 DHFR, asymmetric K32M mutations were generated

using the tandem gene array that encodes Quad3. From studies of these asymmetric mutants, a

revised model for K32 in binding and catalysis is proposed.

EXPERIMENTAL PROCEDURES

Construction of Asymmetric Mutants Construction of asymmetric mutations in our tandem

array is straightforward as the gene copies are separated by unique restriction enzyme sites. We

mutagenize each gene copy separately and then reconstruct the tandem array. This process has

been previously described for production of asymmetric Q67H mutations (6). All K32M

mutants were verified by DNA sequencing.

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Growth and Purification E.coli STBLII cells are able to maintain the tandem gene array without

recombination (14). Cells containing the desired clone were grown in TB media at 30°C for 60

hours in the presence of only 200µg/ml ampicillin, since introduction of trimethoprim into the

growth media results in random mutations within the gene sequences. DNA from each of the 12

liters for the K32M:1+3 and K32M:1+4 mutants was isolated and sequenced to verify that each

culture still possessed the appropriate mutations. Cells were lysed and protein purified as

described previously (6). During the purification process, 0.1g/L polyethylene glycol 3350

(PEG) was added to all solutions since this addition minimizes aggregation (15) and leads to

higher protein yields. Mass spectrometry (intact mass by electrospray-ion trap (4)) was utilized

to verify the K32M:1+2 mutant protein had the correct molecular mass.

pH Titrations Wild type R67 DHFR is a homotetramer that undergoes a pH dependent

dissociation to 2 dimers (16). This process can be monitored by fluorescence as symmetry

related tryptophan 38 residues occur at the two symmetry related dimer-dimer interfaces (17).

At pH 8, W38 is buried (tetramer), while at pH 5 it is exposed to solvent (dimer). The pH

dependence of dissociation arises from protonation of symmetry related H62 residues that also

occur at the dimer-dimer interfaces. While Quad3 cannot dissociate due to the linker sequences

tethering each gene copy product, it can undergo a transition from a "closed" form (active

conformation, pH 8) to an "open" form (inactive, pH 4) (5,6). The effects of pH on Quad3 were

assessed in varying salt conditions at room temperature. The intensity averaged emission

wavelength, <λ>, for each emission spectrum was calculated according to Royer et al. (18). The

pH profiles were fit to a simple ionization equation and normalized according to Bradrick et al.

(5).

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Circular Dichroism Circular dichroism (CD) spectra were recorded at 22°C in 10 mM potassium

phosphate buffer (pH 8) with 10 µM Quad3, K32M:1+2, K32M:1+3, and K32M:1+4 mutant

R67 DHFRs using an Aviv Model 202 series circular dichroism spectrometer as previously

described (4).

Steady-State Kinetics with Mutants The steady-state kinetic behavior of each mutant was

monitored as previously described (19). Experiments were performed at 30°C in either 50 mM

MES + 100 mM Tris, + 50 mM acetic acid (MTH) polybuffer (20) or TE buffer (10 mM Tris, 1

mM EDTA, pH 7) in the presence of various concentrations of NaCl to adjust the ionic strength.

At least five subsaturating concentrations of NADPH and DHF were used to measure activity.

Global fitting of the data to an equation describing the bi-substrate kinetic reaction of R67 DHFR

used a non-linear subroutine of SAS (version 8.2, (6)). The NLINEK macro for use in SAS is

available on the internet at http://animalscience.ag.utk.edu/faculty/saxton/software.htm. The

extinction coefficient for the R67 DHFR reaction at 340 nm is 12,300 L mol-1cm-1 (21). Ligand

concentrations were calculated using an extinction coefficient of 28,000 L mol-1 cm-1 at 282 nm

for DHF (22) and 6220 L mol-1cm-1 at 340 nm for NADPH (23). Kinetic data for the K32M:1+3

mutant were obtained at 360 nm to more accurately assess the Km values. The extinction

coefficient for the DHFR reaction is 5020 L mol-1cm-1 at this wavelength. Extinction coefficients

for NADPH and DHF are 4020 L mol-1cm-1 and 2630 L mol-1cm-1 respectively at 360 nm.

Fluorescence Quenching Binding of NADPH or DHF to 2 µM protein at 4oC was monitored in

TE buffer containing 0.03M NaCl (µ=0.05) using tryptophan fluorescence (24). Data were fit to:

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Fl = Fo – 0.5 Fo [Ptot + Kd + Ltot – [(Ptot + Kd + Ltot)2 – 4 Ptot Ltot)1/2] where Fl is the observed

fluorescence, Ltot is the total ligand concentration, and Ptot, Kd and Fo are variables describing the

number of enzyme binding sites, dissociation constant and fluorescence yield per unit

concentration of enzyme, respectively (25).

Sedimentation velocity Sedimentation velocity experiments were conducted in a Beckman

Optima XL-I ultracentrifuge equipped with both interference and absorbance optics. Protein

samples were dialyzed into TE buffer, pH 7 with 0.03M NaCl added (µ= 0.05) and the dialysate

was used as an optical reference. Varying concentrations from 0.6 µM to 5 µM of each mutated

protein were loaded (400 µl loading volume) into double-sector charcoal-filled epon centerpieces

and sedimentation velocity was carried out at 50,000 rpm at 4°C using an An50 Ti eight-hole

rotor. Sedimentation velocity analysis was performed by direct boundary modeling by solutions

of the Lamm equation using the program Sedfit ((26), see

http://www.analyticalultracentrifugation.com). Partial specific volume, buffer density and

viscosity were determined using the software SEDNTERP (John Philo at AMGEN Corp, see

http://www.jphilo.mailway.com/download.htm).

RESULTS

Nomenclature The following system will be used to name each of the asymmetric mutants. The

residue, residue number, and mutation will be listed first followed by a colon. The asymmetric

location of the particular mutations will be indicated numerically where 1 refers to gene copy 1,

2 refers to gene copy 2, etc. For example, a single K32M:1 mutant was constructed3 where the

mutation was placed in gene copy 1. While three other single mutants could have been

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constructed, the 222 symmetry of wt R67 DHFR predicts they will be equivalent. Three double

mutants were also constructed. The K32M:1+3 mutant contains two mutations: lysine 32 in gene

copy 1 has been mutated to methionine, as has lysine 32 located in gene copy 3. The K32M:1+2

and K32M:1+4 double mutants also have two mutations in their respective gene copies. The

non-equivalency of these constructs can be illustrated using the crystal structure for

homotetrameric R67 DHFR (see Figure 1) where each monomer would correspond to a domain

in Quad3 (2,27). The four domains are labeled in a counter-clockwise fashion where domain 1 is

located at the top, left position. Panels B-D in Figure 1 compare the different topologies of the

three double mutants. For the K32M:1+3 mutant, one side of the active site pore contains two

wild-type K32 residues while the opposite side of the pore contains two mutant K32M residues

(panel C). In contrast, both the K32M:1+2 and K32M:1+4 mutants contain one K32 and one

K32M residue on each side of the pore (panels B and D), although with different geometries

between the mutations. Figure 1E depicts the arrangement of the mutations with respect to a

reverse image of the active site pore and shows our simple model for bound NADPH and folate

derived from docking NADPH into R67 DHFR•FolI (3). Since the K32M mutation destabilizes

homotetrameric R67 DHFR, triple and quadruple mutants were not constructed.

Physical Studies of the Apo Proteins To examine whether the gene quadruplication event

altered the salt sensitivity of the protein, pH titrations of Quad3 were performed in MTH buffer

with various additions of NaCl (Figure 2) (7). The data were fit and the corresponding pKa

values are given in the figure legend. It is apparent that addition of salt slightly stabilizes the

protein. However, there is not a significant difference in the titrations near pH 7, where the

majority of experiments are performed.

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pH titrations of the double mutants were attempted, however asymmetric addition of the

K32M mutation leads to increased aggregation. Visible turbidity usually appeared during

dialysis or during strong mixing/shaking conditions, particularly for concentrated solutions. To

minimize aggregation, the proteins were purified and maintained in buffer solutions containing

0.1g/L PEG 3350 (15). While low concentrations of protein are stable at ≤ 4oC, we were unable

to perform pH titrations of 2µM protein at room temperature.

Since aggregation was a problem with these mutants, we evaluated whether the

oligomerization state of the protein was altered using sedimentation velocity experiments.

Sedimentation velocity profiles for each double mutant at 4oC and at varying concentrations

(≤5µM) were fit using a continuous c(S) distribution model (Sedfit; (26)). The results clearly

demonstrate the presence of a single species for all samples, with S values of 2.4, 2.4, and 3.4 for

the K32M:1+2, K32M:1+3, and K32M:1+4 proteins, respectively. For all fits, conversion to a

continuous c(M) distribution model demonstrated that all three mutants are of the same

molecular mass (~30kDa). (From the gene sequence, the expected molecular mass is ~33.5 kDa,

thus these mutants are monomeric under these conditions.) It is likely that the observed

difference in the S value for K32M:1+4 reflects an alteration in its molecular shape compared to

the K32M:1+2 and K32M:1+3 mutants. Since the K32M mutations in this asymmetric mutant

both occur at what would correspond to one dimer-dimer interface in homotetrameric R67

DHFR, this “interface” appears perturbed in the apo K32M:1+4 mutant (see Figure 1D).

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CD spectra for each of the K32M double mutants at pH 8 are given as supplemental data.

The signals for the K32M:1+2 and K32M:1+3 mutants show some changes with respect to

Quad3, while the spectrum for the K32M:1+4 mutant displays greater differences. These

differences could describe some level of conformational change in the proteins and/or alterations

in the contribution of aromatic residues to the signal. Aromatic residues contribute significantly

to the CD signal in R67 DHFR (17), in accord with general observations by Woody (28). Since

K32 is near W38 (closest approach ~ 4 angstroms), mutation to K32M could potentially affect

the CD signal.

All the above physical studies suggest addition of a K32M mutation(s) results in some

level of structural destabilization. Ocam’s razor suggests the simplest interpretation is alteration

of the structure at what would correspond to the dimer-dimer interface(s) in homotetrameric R67

DHFR. While only limited conformational changes cannot be proven, the steady state kinetic

data below suggest addition of ligand(s) provides sufficient contacts to restore the active site

structure.

Binding of NADPH Monitored By Fluorescence Quenching To monitor how the mutations

affect NADPH binding, a fluorescence quenching approach was used (at 4oC). This technique

only monitors binding at the first tight site, yielding Kd1 (2). The titrations are shown in Figure

3A, and the fitted values are given in Table 1. The tightest Kd1 values are associated with Quad3

and the single mutant, K32M:1, followed by K32M:1+3, and then by the K32M:1+2 and

K32M:1+4 double mutants, which are quite similar in their behavior.

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Another difference between the titrations is the percent quench observed. Similar

quenches are observed for Quad3, K32M:1 as well as the K32M:1+3 mutant, suggesting related

binding modes. Smaller quenches are observed for the K32M:1+2 and K32M:1+4 mutants,

suggesting either NADPH binds differently, or the environment of W38 (which monitors the

titration (17)) is altered by a proximal K32M mutation.

Binding of DHF Monitored By Fluorescence Quenching Binding of DHF to the various

mutants was also performed at 4oC. Particularly for weaker binding mutants, the background

quench associated with addition of DHF becomes substantial, precluding accurate fitting. Thus

the DHF binding data monitored by fluorescence quenching are presented qualitatively in Figure

3B. Binding of DHF to both Quad3 and the single mutant (K32M:1) proteins yields titrations

that are mostly complete upon addition of ~7 µM ligand. An intermediate titration with a

reduced quench is observed for DHF binding to the K32M:1+4 mutant. Finally, the K32M:1+2

and 1+3 double mutants both show minimal quenching as well as much weaker binding.

Alterations in the percent quench could describe either changes in the number of DHF molecules

bound and/or how DHF is juxtaposed near W38. Weaker binding most likely describes lost

interactions between DHF and K32 and/or effects on the positive cooperativity that occurs

during binding of 2 DHF molecules.

A Comparison of Steady-State Kinetic Parameters Since PEG was present in the assays, a test

of its effects on the steady state behavior of Quad3 was performed. As shown in Table 2, no

significant changes were noted. The steady state kinetic parameters for the various mutants are

also listed in Table 2. The behavior of the single mutant protein, K32M:1, is quite similar to that

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of Quad3. More significant changes are observed for the double mutants. The K32M:1+2

mutant displays a 4-fold increase in Km (NADPH), a 2-fold increase in Km (DHF), and a 4-fold

decrease in kcat as compared to Quad3. The K32M:1+4 mutant possesses a similar Km (NADPH) to

Quad3 with a 1.6 fold decrease in the affinity for DHF. However, kcat is decreased 8-fold by this

mutation. Finally, the K32M:1+3 mutant displays the most obvious changes as its Km (NADPH) is

≥160 µΜ, Km (DHF) is ≥ 330 µM and kcat is ≥ 3.6 s-1. These kinetic values are presented as lower

limits due to the large elevation in Km values for both substrate and cofactor coupled with the

absorbance limit of the spectrophotometer. This mutant (1+3, where half the pore contains wt

K32 residues and the other half pore contains K32M mutations) clearly displays at least a 4.5

fold enhancement of the kcat value compared to Quad3.

While the apo structure of the K32M:1+4 mutant (see Figure 1D) appears perturbed by

CD and sedimentation velocity measurements, addition of ligands appears to provide a

reasonable level of compensation since this mutant is as active as the K32M:1+2 mutant. Most

likely binding of NADPH and DHF provides sufficient binding contacts to restore the necessary

structure for activity.

Salt Effects on Kinetic Behavior To evaluate the ionic strength dependence of these steady state

kinetic parameters, salt effects were analyzed for the double mutant series. Data are shown in

Figure 4 and the slopes of the plots are listed in Table 3. For both the K32M:1+2 and K32M:1+4

mutants, kcat is only minimally affected by salt concentration while Km (NADPH) and Km (DHF)

increase with increasing ionic strength. The kinetic behavior for these two double mutants (with

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single K32M mutations in each half of the pore) is similar, suggesting R67 DHFR can

accommodate several topologies of bound ligands.

Finally, we attempted to determine if kinetic values could be obtained for the K32M:1+3

mutant in TE buffer plus 0.03M NaCl, as smaller Km values are usually observed in lower salt

conditions. For this buffer, it appeared that Km (NADPH) could be bracketed, but Km (DHF) could

not. Since the resulting values are linked, they provide only a lower limit and are: Km (DHF) ≥ 490

µM, Km (NADPH) ≥ 60 µM, and kcat ≥ 4.9 s-1.

DISCUSSION

The simplest model of catalysis in R67 DHFR proposes that DHF occupies half the pore

and cofactor the other half. The pteridine ring of DHF and the nicotinamide ring of NADPH

encounter each other at the center of the pore (29) where the reaction occurs. From a comparison

of Kd1 values, a strong preference exists for NADPH to bind first (2). It would presumably

interact with the tightest binding site available. When DHF then binds to the enzyme•NADPH

complex, it is forced into the other half of the pore, which could lack one to two K32 contacts in

this asymmetric mutant series. Binding models for the various mutants are schematized in

Figure 5 and will be discussed below.

Does K32 play a direct role in binding NADPH? Docking of NADPH into R67 DHFR•folate

predicts an ionic interaction between K32 and the 2’phosphate of NADPH (see Figure 1E) (3).

While the pyrophosphate bridge is docked further away from a symmetry related K32 residue,

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sidechain movement could allow formation of a second ionic interaction. Experimental evidence

supporting a second ionic interaction comes from our previous salt effect data (7) where binding

of NADPH to wild type (homotetrameric) R67 DHFR was found to be salt sensitive and a log-

log plot of ionic strength vs. Kd1 yielded a slope of 2. The slopes of these types of plots have

previously been taken to describe Z, the number of ionic interactions involved in binding

(11,12,30).

To evaluate the above model where 1-2 ion pairs are predicted to occur between NADPH

and symmetry related K32 residues, asymmetric mutants were used. A single K32M mutation

did not alter the Kd1 associated with NADPH binding. In the double mutant series, the

K32M:1+3 mutant shows the least effect, with an ~ 5 fold increase in Kd1 and a similar percent

quench as Quad3. The K32M:1+2 and K32M:1+4 double mutants show ~10 fold weaker

binding and reduced quenching of protein fluorescence, suggesting more perturbations are

associated with NADPH binding. Effects on binding are likely to include loss of an interaction

between K32 and NADPH as well as any alterations in the electrostatic potential, and/or in κ, the

orientational effect (refer to Figure 5 for models). (Effects on κ, the orientational effect in the

modified Smoluchowski equation describing diffusion, would be expected to decrease kon (31).)

When NADPH binds to the K32M:1+3 double mutant, it may bind to either the half pore

possessing wildtype K32 residues or the half pore containing mutant K32M residues (see Figure

5). Presumably, binding to the wildtype half pore would involve fewer perturbations and result

in minimal effects on Kd1. This expectation is consistent with the observed dissociation constant.

However as mutations are added to each half pore (as in the K32M:1+2 and K32M:1+4 double

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mutants), larger effects would be expected if an ionic interaction is lost upon NADPH binding.

Surprisingly, the observed effect on Kd1 for the K32M:1+2 or K32M:1+4 mutants is small (~10

fold when compared to Quad3 and <2 fold when compared to K32M:1+3), suggesting that either

R67 DHFR utilizes only one ionic interaction in binding NADPH or that if a second ionic

interaction occurs, it is quite weak.

When the Kd1 (NADPH) values are compared with the Km (NADPH) values (TE with 0.03M

NaCl pH 7.0), it is clear that some Km values must include kinetic terms. For example, while the

Kd1 (NADPH) for the K32M:1+3 double mutant is 5.2 µM, the corresponding Km (NADPH) value is ≥

60 µM.

When kcat/Km (NADPH) values are compared, the double mutants become equivalent (~0.01-

0.02 s-1µM-1) and are approximately 10-20 fold less efficient than Quad3. The observation of

similar kcat/Km (NADPH) values for all double mutants over a ≥ 36 fold kcat range indicates some

degree of non-productive binding likely occurs. Non-productive binding dictates that kcat/Km

values remain constant while additional binding modes lead to apparently tighter binding.

However since only a fraction of these binding modes is productive, kcat is also decreased in

parallel (32). Non-productive binding in R67 DHFR would not be surprising due to its 222

symmetry. Since the highest kcat and Km values occur for the K32M:1+3 double mutant, this

protein likely possesses the most productive binding mode (in the double mutant series). In other

words, loss of ionic contacts in one half of the pore in this mutant (see Figure 5) leads to more

productive binding. Conversely, non-productive binding likely describes ionic contacts in one

half of the pore between K32 and DHF or NADPH.

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What is the role of K32 in binding DHF? From binary complex binding studies (Figure 3B),

K32 clearly plays a role in binding DHF. Loss of one K32 residue is well tolerated, but loss of

two K32 residues affects both the percent quench as well as binding affinity. Changes in

affinity must remain qualitative as the curves were not fit.

If our cartoon describing the ternary complex in Figure 5 (also see Figure 1E)

approximates productive ground state binding, then minimal effects on kcat and Km would be

expected for the asymmetric mutant that matches the (predicted) productive ternary complex

topology (K32M:1+2), with larger effects on the non-preferred topology (K32M:1+4). However

we find that the 1+2 and 1+4 double mutants have similar kinetic behavior (<3 fold differences

in both kcat and Km values). This is a highly unusual result and we conclude that both topologies

allow comparable interactions between K32 and the glu tail of DHF. This behavior correlates

with the disorder observed for the glu tail of bound folate in the crystal structure (1) as well as

NMR studies that find the glu tail is mobile (29).

These data present a conundrum, how can the DHF tail be mobile, yet nearby residues

affect its’ binding? Perhaps the tail could maintain solvent separated ion pairs (SSIP) with both

symmetry related K32 residues. SSIP are a common phenomenon in small molecules (33-35) &

may play a role in enzymes (36,37). Solvent separation could minimize the desolvation penalty

associated with ion pair formation (38). Also some host-guest studies indicate tight binding can

be associated with high ligand mobility in the cavity (39-41).

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From our binding model (2), NADPH binds first and interacts with the wt half of the

pore, while DHF is forced into the mutant half of the pore lacking K32 contacts (see Figure 5).

This model is supported by the asymmetric mutant data garnered in this study as Km (DHF) is

weakened >40 fold in the K32M:1+3 mutant when compared to Quad3. DHF is unlikely to bind

first as our fluorescence quenching studies do not indicate appreciable formation of a

K32M:1+3•DHF complex (up to 27µM DHF), while NADPH binds reasonably well to this

mutant under identical conditions (Figure 3). Since interligand cooperativity is important to R67

DHFR function (2,9), weaker binding of DHF affects NADPH Km values as well.

What is the role of K32 in catalysis? In wt R67 DHFR, an increase in kcat is associated with

increasing salt conditions (7). An increase in kcat also occurs in the K32M:1+3 mutant with a ≥

4.5 fold increase over Quad3 and a ≥ 18-fold increase over the other double mutants. Thus, this

mutant, which appears to have lost its potential for an ionic contact(s) in one half the pore due to

the substitution of both K32 residues, to some degree mimics the effects of salt on kcat for wt R67

DHFR. These data indicate that charge neutralization facilitates formation of the transition state.

What is the salt sensitive interaction monitored by kcat? Since salt sensitivity for kcat

remains when NADH is used as the alternate cofactor in wt R67 DHFR, the 2’phosphate moiety

is not involved (7). Either the pyrophosphate bridge of NADPH or the pABA-glu tail of DHF

remain as possible candidates. Our results for the K32M:1+3 double mutant suggest the salt

sensitive interaction may be with the pABA-glu tail of DHF. A weak, salt sensitive interaction

can be envisioned as beneficial as it could be more readily broken. Also the ability of the

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K32M:1+2 and 1+4 double mutants to tolerate various positions of the pABA-glu tail could

correlate with the requirement to break a salt bridge with K32 to reach the transition state.

Another obvious effect of the asymmetric K32M mutations is on the slopes of log-log

plots of ionic strength vs. kcat. The slope for wt R67 DHFR is 0.9 while the slope for the

K32M:1+2 and 1+4 mutants decreases to 0.2. Clearly an interaction with K32 is involved in loss

of some level of salt sensitivity. Since the change in slope is not unitary and some salt sensitivity

remains, does this correspond to loss of a single ionic interaction? This scenario seems unlikely

as some level of non-productive binding appears to have been unmasked in the double mutants.

Since the slopes for kcat/Km remain within error of each other, it seems likely that the decrease in

slope for kcat corresponds to different salt sensitivities associated with the productive and non-

productive binding modes, with the non-productive mode being less susceptible in this ionic

strength range.

Conclusions Three critical observations from our asymmetric K32M mutations that provide

further detail on R67 DHFR function are: 1) a non-productive binding mode has been uncovered

in R67 DHFR as all three double mutants show similar kcat/Km values. The nonproductive

binding mode appears to correlate with interactions between K32 and the pABA-glu tail of DHF.

2) Similar kcat and Km values for the K32M:1+2 and K32M:1+4 double mutants are observed and

indicate alternate pABA-glu tail positions of DHF are tolerated by the enzyme. 3) The

K32M:1+3 double mutant shows an enhanced kcat coupled with weaker binding of DHF. This

result indicates charge neutralization enhances transition state formation, albeit at the expense of

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ground state binding. These opposing trends arise from the 222 symmetry of the enzyme, which

dictates that each residue must serve numerous roles, none of which are likely to be optimized.

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FOOTNOTES

1. Abbreviations: DHFR, dihydrofolate reductase; wt, wild type; TMP, trimethoprim; DHF,

dihydrofolate, NADP(+/H), nicotinamide adenine dinucleotide phosphate (oxidized/reduced);

NMNH, reduced nicotinamide mononucleotide; MTH buffer, 50 mM MES + 100 mM Tris, + 50

mM acetic acid polybuffer; µ, ionic strength; TE, 10 mM Tris + 1 mM EDTA buffer; ITC,

isothermal titration calorimetry; CD, circular dichroism; pABA-glu, the para-aminobenzoic acid-

glutamate tail of dihydrofolate; and Quad3, the protein product of the quadruplicated R67 DHFR

gene. Mutant enzymes containing amino acid substitutions are described by the wild type

residue and numbered position in the sequence, followed by the amino acid substitution. For

example, K32M R67 DHFR describes the lys32 → met mutant.

2. The amino acids in the first monomer (A) are labeled 1-78; those in the second monomer (B),

101-178; those in the third monomer (C), 201-278; and those in the fourth monomer (D), 301-

378. For brevity, when a single residue is mentioned, all four symmetry related residues are

implied. The monomer arrangement going clockwise in the crystal structure (1VIE.pdb) is

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ABDC. To minimize confusion in the quadruplicated gene construct, we have re-labeled the

monomers 1234 (= ABCD) going clockwise.

3. For simplicity as well as to provide consistent nomenclature with other asymmetric mutants

(e.g. (6) and Stinnett et al., companion paper), we use the K32M:1, K32M:1+2 or 1+3 or 1+4

nomenclature. However the single mutation was actually placed in the second gene copy.

Because of the 222 symmetry, the K32M:2 construct should be equivalent to a K32M:1

construct. The double mutants actually constructed were K32M:3+4, K32M:2+4, and

K32M:2+3 (which should be equivalent to the K32M:1+2, K32M:1+3 and K32M:1+4 constructs

from the 222 symmetry). These constructs were made as we were concerned that two K32M

mutations at what corresponds to a dimer-dimer interface in wildtype (wt) R67 DHFR could be

destabilizing. Thus we placed the two K32M mutations into gene copies 2 and 3. Our reasoning

for this placement was to allow the wild type (wt) sequences in copies 1 and 4 to interact at the

N- and C-termini and provide contacts to hold the domains together.

ACKNOWLEDGEMENTS

We thank Cynthia Peterson for her assistance with the sedimentation velocity experiments and

for critical reading of the manuscript. Special thanks to Nathan C. VerBerkmoes and Robert L.

Hettich at the Organic and Biological Mass Spectrometry group at Oak Ridge National

Laboratory for intact mass spectrometry analysis of the K32M:1+2 mutant.

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FIGURE LEGENDS

Figure 1. The structure of R67 DHFR (1VIE in the protein data bank). Each monomer in wt

homotetrameric R67 DHFR corresponds to a domain in Quad3. Monomer 1 is colored red,

monomer 2 is yellow, monomer 3 is magenta, and monomer 4 is green. The K32 positions are

shown using a CPK representation. Panel A shows a ribbon drawing of the homotetramer

looking end on with the active site pore in the center. The N-termini point to the left and right

sides of the image while the C-termini point up and down. The protein in this crystal form was

obtained by chymotrypsin treatment such that 16 amino acids were cleaved off the N-terminus

(1,27). Panels B-D, the ribbon structures are related to panel A by a 90° rotation along the y-

axis. The expected positions of the mutations are shown in white whereas wild-type K32

residues are indicated in color. Panel B illustrates the K32M:1+2 double mutant, its mutations

lie across the pore from one another in a diagonal orientation (i.e. across the monomer-monomer

interface (red and yellow). Panel C depicts the K32M:1+3 double mutant; both mutations lie in

the same half- pore. Panel D describes the K32M:1+4 mutant; it possesses mutations on

opposite sides of the pore, arranged consecutively (i.e. across along the dimer-dimer interface

(red and green). Panel E shows a reverse image of the active site pore generated by DOCK

(3,42,43). The orientation of this sphere cluster (with respect to the homotetramer) corresponds

to panels B-D. Each white point corresponds to a potential atom position used by the docking

program. The position of a docked NADPH molecule that meets the NMR constraints

(29,44,45) is shown in cyan and the position of the highest scoring docked folate molecule is

shown in orange. The positions of the 4 symmetry related K32 residues in the surrounding

protein are colored and numbered as described above. The rest of the protein is not shown for

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simplicity. Only 1 out of 4 symmetry related binding modes is shown. The other binding modes

can be generated by 180o rotations around the x, y and z axes.

Figure 2. pH titration curves describing the behavior of Quad3 in the presence of various

concentrations of NaCl. This experiment examines salt effects on the transition from a “closed”

(pH 6-8) to an “open” (pH 4-5) form. Titration experiments were performed in the presence of

MTH buffer ( , ), MTH buffer containing 0.2M NaCl ( , ), MTH containing 0.5M

NaCl ( , ), and MTH containing 0.75M NaCl ( , ). Best fits for these curves yield

pKa values of 5.9 ± 0.01, 6.0 ± 0.01, 5.7 ± 0.01 and 5.6 ± 0.02, respectively.

Figure 3. Fluorescence quenching of Quad3 variants by NADPH or DHF. Panels A and B

show the NADPH and DHF titrations respectively. The data were normalized to allow ready

comparison. The titrations generated for Quad3 (hexagons, fit by solid line), K32M:1 (∇ points

and dot-dash-dash line), K32M:1+2 ( points and a dot-dash line), K32M:1+3 ( points and a

dashed line), and K32M:1+4 mutants ( points and a dotted line) are shown. Best-fit values for

the NADPH titrations are given in Table 1. The DHF titrations were not fit as the baseline

quench for DHF varied and became substantial for the weaker binding mutants.

Figure 4. Log-log plots of each kinetic parameter vs. ionic strength generated for the K32M:1+2

( points) and K32M:1+4 ( points) double mutants. Steady-state kinetics were performed in

TE buffer pH 7 (30°C) with various salt concentrations added to adjust the ionic strength. There

is a linear correlation between each parameter and ionic strength. Slopes of the various plots are

given in Table 3.

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Figure 5. Cartoons describing how NADPH and DHF may bind to the various mutants. The

dumbbell shaped outline represents the active site pore. Bound NADPH is shown by a light gray

patch on the right and bound DHF is depicted by a darker gray area on the left. The 2’phosphate

and pyrophosphate groups of NADPH are represented by a circle and square and the glu tail of

DHF by a diamond. For Quad3, only one of four possible symmetry related binding modes is

shown. The positions of the four symmetry related K32 residues in the surrounding protein are

also shown in a cartoon fashion. They are numbered 32, 132, 232 and 332 and are colored black,

dark gray, light gray and dotted respectively. The rest of the protein is not shown for simplicity.

Potential ionic interactions are indicated by the juxtaposition of the K32 residues with the circle,

square or diamond. As residues are mutated to methionine, the K32 patches are removed,

indicating the loss of any potential contact. As mutations are added, the occupancy of binding

sites containing K32M substitutions is expected to decrease. For example, as NADPH has a

clear preference to bind before DHF (2), we have placed it in the least perturbed half pore to

maintain the maximal number of contacts, see K32M:1 and K32M:1+3. When K32M mutations

are added to each half pore, the preferred binding configuration is expected to be a function of

the strongest interaction(s). For example, if a preference exists for an ionic interaction between

the 2’phosphate of NADPH and K32, then that site will be occupied (see K32M:1+2 as well as

the K32M:1+4 model where the ligands are flipped and occupy a symmetry related site).

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Table 1. Fluorescence quenching of Quad3 and the K32M mutants by NADPH. These

experiments were performed at 4°C in TE buffer, pH 7 in the presence of 0.03M NaCl (µ=0.05)

and 0.1g/L PEG.

Species Kd1 (µM)

Quad3 1.0 ± 0.1

K32M:1 1.2 ± 0.04

K32M:1+2 9.7 ± 0.7

K32M:1+3 5.2 ± 0.5

K32M:1+4 8.4 ± 0.4

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Table 2. Steady-state kinetics were performed with the K32M asymmetric single and double

mutants in MTH buffer, pH 7 at 30°C. Data were fit using a non-linear global option in SAS to

an equation describing bi-substrate kinetics.

Protein Km (NADPH) (µM)

Km (DHF) (µM)

kcat (s-1)

kcat / Km (NADPH) (s-1 µM-1)

kcat / Km (DHF) (s-1 µM-1)

Wild Type R67 DHFRa

3.0 ± 0.06 5.8 ± 0.02 1.3 ± 0.1 0.43 ± 0.03 0.22 ± 0.01

Quad 3b

4.4 ± 0.4 6.7 ± 0.4 0.80 ± 0.02 0.18 ± 0.02 0.12 ± 0.01

Quad3 + PEG

3.9 ± 0.2 7.6 ± 0.4 0.85 ± 0.03 0.22 ± 0.02 0.11 ± 0.01

K32M:1

3.6 ± 0.2 6.0 ± 0.3 0.56 ± 0.01 0.16 ± 0.01 0.093 ± 0.006

K32M:1+2

17.1 ± 0.1 14.4 ± 0.1 0.20 ± 0.01 0.012 ± 0.001 0.014 ± 0.001

K32M:1+3 ≥ 145 ≥ 400 ≥ 3.7

0.026c 0.009c

K32M:1+4 5.4 ± 0.2 10.5 ± 0.3 0.10 ± 0.01 0.019 ± 0.003 0.010 ± 0.001

a from (27).

b from (6). c presented without errors as saturation with neither substrate could be obtained.

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Table 3. Slopes of log-log plots of various kinetic parameters vs. ionic strength for the

K32M:1+2 and K32M:1+4 mutants compared to wt R67 DHFR.

DHFR

Species Slope kcat/Km

(NADPH)

Slope kcat/Km (DHF)

Slope kcat Slope Km (NADPH)

Slope Km (DHF)

wt R67 DHFRa

-0.6 ± 0.09 -0.9 ± 0.03 0.9 ± 0.08 1.5 ± 0.1 1.8 ± 0.2

K32M:1+2 -1.1 ± 0.13 -0.72 ± 0.16 0.2 ± 0.05 1.3 ± 0.02 0.9 ± 0.02

K32M:1+4 -1.2 ± 0.1 -1.2 ± 0.2 0.2 ± 0.05 1.4 ± 0.04 1.4 ± 0.09

a from (7).

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Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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35

igure 5.

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Circular dichroism spectra for Quad3 and each of the K32M double mutants at pH 8. The spectrum for Quad3 is given by the solid line; that for K32M:1+2 by a dashed line; K32M:1+3 by a dotted line; and K32M:1+4 by a dash-dot-dot line.


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HowellStephanie N. Hicks, R. Derike Smiley, Lori G. Stinnett, Kenneth H. Minor and Elizabeth E.Role of K32 residues in R67 dihydrofolate reductase probed by asymmetric mutations

published online August 27, 2004J. Biol. Chem.

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