Proton Magnetic Resonance Studies on Escherichia coZi ...

Vol. 254, No 17, Issue of September 10. pp. 8143-8152, 1979 Prmtedin U.S.A.

Proton Magnetic Resonance Studies on Escherichia coZi Dihydrofolate Reductase ASSIGNMENT OF HISTIDINE C-2 PROTONS IN BINARY COMPLEXES WITH FOLATES ON THE BASIS OF THE CRYSTAL STRUCTURE WITH METHOTREXATE AND ON CHEMICAL MODIFICATIONS*

(Received for publication, March 19, 1979)

Martin Poe and Karst Hoogsteen

From the Merck Znstitute for Therapeutic Research, Rahway, New Jersey 07065

David A. Matthews

From the Department of Chemistry, University of California at San Diego, La Jolla, California 92093

The effects of pH upon the C-2 resonances of the 5 histidine residues of Escherichia coli MB 1428 dihydrofolate reductase in binary complexes with methotrexate, aminopterin, folate, methopterin, and trimethoprim were studied by 300-MHz ‘H nmr spectroscopy. Three of the five histidine residues, labeled 1, 2, and 3, exhibited similar pK’ values and chemical shifts for their C-2 protons in the five binary complexes. One histidine, 4, was quite different in the folate complex and the last histidine, 5, was quite different in the trimethoprim complex. For all five binary complexes, each histidine had a pK’ which was significantly different from the other 4 histidines of that complex. Titra- tion of the binary methotrexate complex of a 5,5’-dithiobis(2-nitrobenzoate)-modified enzyme showed that 2 histidines were not perturbed by this modification of Cys 152, and that the alkaline form of histidine 2, the acid form of histidine 4, and, to a lesser extent, the acid form of histidine 3 were slightly perturbed. Titration of the binary methotrexate complex of a N-bromosuccinimide-modified enzyme demonstrated that this modification slightly affected all of the histidines and dras- tically affected histidine 5. Histidines 3 and 5 of the binary methotrexate complex reacted rapidly with the histidine-specific reagent, ethoxyformic anhydride, while histidines 2 and 4 reacted at a moderate rate and histidine 1 reacted slowly if at all.

The local electrostatic environments of the 5 histidine residues as deduced from the crystal structure of the binary complex of the enzyme with methotrexate (Mat- thews, D. A., Alden, R. A., Bolin, J. T., Freer, S. T., Hamlin, R., Xuong, N., Kraut, J., Poe, M., Williams, M. N., and Hoogsteen, K. (1977) Science 197,594-597) were used as the basis for proposed assignments of the five histidine C-2 nmr resonances. The assignments were: 1, pK’ 7.9 to 8.2, His 124; 2, pK’ 7.2 to 7.4, His 141; 3, pK’ 6.5 to 6.7, His 149; 4, pK’ 5.7 to 6.3, His 114; and 5, pK’ 5.2 to 5.9, His 45. The effect of the chemical modifications upon the enzyme’s histidine residues were consistent with the assignments, but no direct chemical evidence in support of the assignments was obtained. It was proposed that, since the crystallographic data provided consistent assignments of the histidine nmr data for both native and chemically modified enzyme,

* This work was supported in part by Research Grant CA 17374 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 USC!. Section 1734 solely to indicate this fact.

the local environment of each of the 5 histidine residues was similar in the crystal and in solution.

The enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofo- 1ate:NADP’ oxidoreductase EC1.5.1.3) is the molecular site of action for several drugs used in cancer chemotherapy (1) and in the treatment of bacterial and parasitic infections (2). The recent solutions of the x-ray crystal structures of the binary methotrexate complex of Escherichia coli MB 1428 dihydrofolate reductase by Matthews et al. (3) and of the ternary methotrexate plus NADPH complex of Lactobacillus casei dihydrofolate reductase by Matthews et al. (4) have provided detailed insight into the geometry and local environment of the binding site for these drugs.

The crystal structure provided information about the local environment of the histidine residue in E. coli dihydrofolate reductase. Histidine residues have been shown by chemical modification studies (5, 6) to be important in maintaining the active configuration of E. coli dihydrofolate reductase. We report here ‘H nvr data on the binary complexes of E. coli MB 1428 dihydrofolate reductase with methotrexate, aminopterin, folate, N( lO)-methylfolate, and trimethoprim under conditions comparable to the crystallization conditions, in order to examine the histidine environments in solution. We propose on the basis of the crystal structure (3) an assignment of the five ‘H nmr resonances that correspond to the C-2 protons of the 5 histidine residues of the protein. Also presented are the results of ‘H nmr studies on the binary methotrexate complex of the E. coli reductase modified with N-bromosuccinimide and 5,5’-dithiobis(2-nitrobenzoate). The results on the specifically modified enzyme are readily reconciled with the assignments based on the crystal structure. The results on the modified enzyme are also useful in analysis of the histidine titrations of pyridine nucleotide complexes of the enzyme, to be presented in a forthcoming paper.]

EXPERIMENTAL PROCEDURES

Materials

E. coli MB 1428 dihydrofolate reductase was isolated and purified according to Poe et al. (7) as modified by Williams et al. (8).

Folic acid, dihydrate was purchased from Cycle; methopterin (N(lO)-methylfolate) was a gift of Dr. E. W. Cantrall, Lederle; methotrexate, a gift of Dr. H. B. Wood, Jr., National

’ M. Poe and K. Hoogsteen, manuscript in preparation.

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8144 His Titrations of Dihydrofolate Reductase

Cancer Institute; trimethoprim, a gift of W. B. Gall, Merck; and aminopterin was purchased from Lederle. All were used without further purification and were standardized spectro- photometrically using: folate, at pH 13, ~(365 nm) = 8500 cm-’ Mm’ (9); methopterin, at pH 13, ~(302 nm) = 26,900 cm-’ Mm’

(10); methotrexate, at pH 13, ~(302 nm) = 22,000 cm-’ M-’

(11); trimethoprim, at pH 1, ~(271 nm) = 6070 cm-’ Mm’; and aminopterin, at pH 13, ~(284 nm) = 26,400 cm-’ Me’ (11). The 1:l complexes of E. coli MB 1428 dihydrofolate reductase with the following ligands were standardized spectrophotometri- tally at 280 nm in 0.05 M NaCl, 0.05 M Tris-HCl, pH 7.2, at 25°C using the following extinction coefficients: folate, 59.0 Cm-’ IIIM-‘; methopterin, 60.6 cm-’ rniv-‘; methotrexate, 48.3 Cm-’ ITIM-‘; trimethoprim, 35.5 cm-’ mM-‘; and aminopterin, 49.3 cm ~’ mM-‘. These extinction coefficients were calculated using ~(280 nm) = 32.0 cm-’ mM-’ for the enzyme, the ligands’ ~(280 nm) values were calculated as standardized above, and using the extinction coefficient change upon ligand binding as determined in ultraviolet difference spectra, taken as described before (12).

N-Bromosuccinimide was purchased from Aldrich Chemi- cal Co. and was recrystallized twice from distilled water the day of use. 5,5’-Dithiobis(2-nitrobenzoic acid) and dithiothreitol were purchased from Calbiochem and ethyl oxydiformate (ethoxyformic anhydride) and glutathione were from East- man.

Chemical Modification

The preparation of DTNB’-modified enzyme was based on the work of Williams and Bennett (13), the NBrSuc modification was done based on Williams (5), and the ethoxyformic anhydride modification was based on Greenfield (6).

DTNB Modification-First, 18.7 mg of E. coli MB 1428 dihydrofolate reductase (1.04 pmol) was reacted with 5 mg of dithiothreitol for 30 min at 23°C. The enzyme then was separated from the reducing agent by chromatography on a Sephadex G-75 column (5 x 80 cm) in Buffer A (0.30 M NaCl, 0.05 M Tris-HCl, pH 7.2). The combined enzyme fractions then had 1.2 pmol (0.55 mg) of methotrexate added. Next, 12.68 mg of DTNB in 2 ml of Buffer A were added, and the absorbance at 412 nm of the solution was monitored contin- uously. When the absorbance at 412 nm reached a steady value, the solution was concentrated by ultrafiltration at 50 p.s.i. and 23°C over an Amicon PM-10 membrane to 1.5 ml. Then, 8.5 ml of Buffer B (0.10 M NaCl, 0.02 M bis-Tris, 0.02 M borate, 0.01 M CaCl?, pH 7.2) was added, and the solution was ultrafiltered to 0.50 ml, followed by lyophilization. Com- parison of the absorbance at 412 nm formed during the reaction of the binary enzyme. methotrexate complex with DTNB with the absorbance formed by DTNB reaction with a standardized solution of glutathione in Buffer A showed that 1.08 Cys residues of the enzyme had reacted with DTNB during the modification.

NBrSuc Modification-First, 12 mg of enzyme (0.67 pmol) was modified by addition of about 5:l NBrSuc:enzyme. This resulted in a 40% loss of enzyme activity with essentially no loss in enzyme absorbance at 280 nm. For modification, 13.6 mg of twice-recrystallized N-bromosuccinimide was dissolved to 5 ml in 0.05 M NaCl, 0.05 M Tris-HCl, pH 7.2. Addition of small aliquots of the NBrSuc solution to the enzyme in Buffer A was done according to the procedure described by Williams (5). When sufficient NBrSuc had been added to give a 40%

’ The abbreviations used are: DTNB, 5,5’-dithiobis(2.nitrobenzoate); NBrSuc, N-bromosuccinimide; pH*, direct pH electrode reading in “H>O solution; 6(HA’) and S(A), chemical shift of His C-2 proton resonance for protonated and neutral form of the residue, respectively; bis-Tris, 2,2’-bis(hydroxymethyl)-2,2’,2”-nitriloethanol.

loss of activity, 1 pmol (0.45 mg) of methotrexate was added, and the solution was prepared for nmr spectroscopy as described above for the DTNB sample.

Ethoxyformic Anhydride Modification-This modification was done directly in the nmr tube with 0.50 ml of 0.76 IIlM

binary enzyme.methotrexate (0.38 pmol) at pH* 7.27 and 25°C. The binary enzyme complex was incubated 12 min at 25°C with 0.05% ethoxyformic anhydride (5 ~1 of a 5% w/w solution in C”H&“H20”H) and it took 15 min to obtain the ‘H nmr spectrum.

Crystallographic Coordinates

Coordinates used in this discussion and in Figs. 8 to 12 are based on a 2.5-A model for the reductase. methotrexate binary complex reported by Matthews et al. (3).

Usually, the electron density for a histidine side chain in a 2.5-A resolution map can be fit equally well by either of two side chain conformations differing in rotations about C&, by 180”. At this resolution, nitrogens cannot be distinguished from carbons, so although the density envelope for the imidazole ring may be well defined, there are two alternative choices for the orientation of the ring within that density. I f one of the two choices is preferable in that one or both ring nitrogen atoms are in a position to hydrogen bond with adjacent backbone or side chain atoms, it is assumed that the conformation permitting hydrogen bond formation will be favored.

The binary methotrexate complex of E. coli dihydrofolate reductase crystallizes with two independent molecules in the crystallographic asymmetric unit (3). Thus, there are two independent determinations of the conformation and local environment for each of the 5 histidine residues. When corresponding side chains in the two independent molecules have the same conformations and the same positions relative to neighboring side chain and main chain atoms, it strongly suggests that the observed tertiary structure has been unaf- fected by crystallization. This is found to be the case for each histidine with a single exception to be discussed below.

NMR Spectra

All ‘H nmr spectra were obtained on a Varian SC-300 MHz spectrometer at 25 + 0.5”C. Typically, 500 to 2000 0.75-s transients were accumulated in the Fourier transform mode on a 0.3 to 1.5 mM solution; an exponential weighting of the free induction decays with a time constant of -0.4 s was used for sensitivity enhancement. Pulses of 5 ys (90” pulse) were used. Spectra were internally referenced to 2,2,3,3-[“H4]tri- methylsilylpropionic acid (Merck Sharpe & Dohme of Can- ada) in units of Hertz or parts per million with downfield shifts positive. pH measurements in ‘HZ0 are direct electrode readings; pH changes and measurements were made as before (14). pH* is the direct electrode reading, as defined by Markley (15). Unless pH* measurements before and after spectral accumulation agreed within +0.04 unit, the nmr data were discarded.

For histidine C-2 resonances, the pK’ and S(HA’), chemical shift at acid pH, were determined by visual fitting of pH titration data to a standard histidine C-2 titration curve with a chemical shift change of 300 Hz upon protonation, with the proportion of acid (HA’) and neutral (A) forms given by pH = pK’ + log([A]/[HA’]) where [HA’] and [A] are the con- centration of acid and neutral forms, respectively.

Solutions of enzyme. ligand complexes were prepared essentially as before (14), save that the final dialysis was against 100 volumes Buffer B. The chemically modified enzyme solutions were prepared as described in the preceding section.


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His Titrations of Dihydrofolate Reductase 8145

RESULTS

Summarized in Fig. 1 are the structures of four heterocyclic compounds which bind tightly to dihydrofolate reductase; the numbering system is that of the IUPAC-IUB for folate (16). Trimethoprim is 3’,4’,5’-trimethoxybenzyl-2,4-diaminopyrim- idine. The binding constant for methotrexate to E. coli MB 1428 dihydrofolate reductase in 0.05 M NaCl, 0.05 M Tris-HCl, pH 7.2, is 4 nM (8), that for folate is 3 pM (17), and that for methopterin is 3 f 1 PM.:’ The competitive inhibitor aminopterin in the same buffer has an inhibition constant of 3.4 nM,

while the inhibition constant for trimethoprim is 3.6 + 0.2 nM

(18). Thus, at the concentrations of enzyme used for nmr studies, 0.3 mM and up, the enzyme is fully complexed.

The low field portion of the ‘H nmr spectra of 1:l complexes of E. coli MB 1428 dihydrofolate reductase with folate, methopterin, aminopterin, and methotrexate are shown in Fig. 2. These spectra were each taken at pH* 7.2; the resonances labeled 1 to 5 are resonances that exhibit the characteristic dependence of chemical shift upon pH that suggest they correspond to histidine C-2 protons (19). The numerical des- ignation of the titrating histidines correspond to their pK’ values, with histidine 1 having the highest pK’ and histidine 5, the lowest pK’. There are a number of broad resonances in the 2200- to 3000-Hz region which appear to exchange slowly for deuterium; these probably are attributable to protons on buried amides. This ‘H nmr resonance region is only slightly downfield of the 2080- to 2280-Hz region where the protons bound to aromatic carbons of tyrosine, tryptophan, and phen- ylalanine are found in extended, random-coil polypeptides (20). The narrow, nonexchangeable resonances in Fig. 2 that exhibit slight or no pH dependence in their chemical shift probably correspond to downfield-shifted aromatic protons.

A plot of chemical shift uersus pH* for the resonances in Fig. 2 is shown in Fig. 3. The curves labeled 1 to 5 in Fig. 3 correspond to the resonances in Fig. 2 with the same label. The labeled solid lines connecting the data points are standard histidine C-2 titration curves, except curve 2 for the enzyme. methotrexate complex, which is fit to a standard curve with a chemical shift difference of 371 Hz between acid and neutral forms. A standard curve is herein defined as a plot of chemical shift uersus pH* with chemical shift 6 given by S = (S(HA’)[HA’] + S(A)[A])/([A] + [HA+]) where S(HA’) and 6(A) are the chemical shifts of the protonated and neutral ionic forms, respectively, with a &HA+) minus 6(A) value of 300 Hz. The area of resonance 3 in enzyme.folate at pH* 6.81 corresponded to 0.95 f 0.25 protons when compared to the area under the aromatic resonances between 1877 and 2321 Hz which were assumed to exhibit resonances for 79 protons (30 protons on the 6 Phe residues, 25 on the 5 Trp residues, 16 on the 4 Tyr residues, 2 on the p-aminobenzoyl moiety of the folates, 1 His C-2, and 5 His C-4 protons). At pH* values near their pK’, several of the histidine C-2 resonances were broad- ened; these resonances were narrow at extremes of pH*. There were no large differences in the rates of deuterium exchange at C-2 for the 5 histidine residues under conditions where the enzyme was stable.

Summarized in Table I are the pK’ values for the 5 histidine residues of five binary complexes of E. coli MB 1428 dihydrofolate reductase. The standard error in these pK’ values is estimated to be +-0.10 unit. The pK’ values were uncorrected for the effect of deuterium upon a pH electrode or upon proton dissociation constants; for histidine residues these effects roughly cancel one another (19). Summarized in Table II are the chemical shifts for the acid form of the histidine residues, 6(HA+); these were extrapolated values for all but resonance

,’ M. Poe, unpublished data.

Rl

\ P”/

RI ‘72 Compound

1. NH2 CH3 Methotrexate

2. N”z H Aminopterin

3. OH CH3 Methopterin

4. OH H Folic acid

FIG. 1. Structural formulas and numbering system for four folates.

METHOPTERIN

I I , I L I i I I

3000 2600 2200

HZ

FIG. 2. Lowest field portion of 300-MHz ‘H nmr spectra of binary complexes of E. coli MB 1428 dihydrofolate reductase. The ligand is identified above each spectrum. Enzyme, 0.4 to 0.8 mM

at 25°C and at pH* 7.24 (folate), 7.15 (methopterin), 7.21 (aminop- t&n), and 7.25 (methotrexate).

1. The chemical shift of the neutral forms were 300 Hz less, except for resonance 2 in the methotrexate complex, which was 371 Hz less.

It was fortunate for making assignments that, in most of the binary complexes whose histidine titrations are summarized in Tables I and II, all 5 histidine residues had distinct pK’ values. It was also helpful in making assignments that there was a good deal of correspondence between the &HA’) and pK’ values of a particular histidine in the five complexes. Histidines 1, 2, and 3 had the same pK’ within +0.3 unit and the same 6(HA+) within ?32 Hz in all five complexes. How- ever, histidine 2 in the methotrexate complex had a signifi-


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1 Trlmethoprlm

lop.\, I -.T.-.-.-.--, -.-:1:-.!i++.

‘p ‘a ,\ -.-.L.-.-:\J+. \ ’ o- .-.-.-.-. A.-. i

\ \z -*-,- l \*.. \ -oq;:v:G o h 4 \ k.-.-

TABLE I

Histidine pK values for E. coli dihydrofolate reductase complexes Values were obtained at 25°C in 0.10 M NaCI, 0.02 M borate, 0.02

M bis-Tris. 0.01 M CaCIl.

Ligand Histi- Assign- dine ment Metho- Aminop-

trexate terin Folate Methop- m;;;o- terin orim

1 124 8.0 8.1 8.2 8.1 7.9 2 141 7.2 7.4 7.2 7.2 7.4 3 149 6.6 6.6 6.6 6.7 6.5 4 114 6.2 6.3 5.7 -6.0 6.1 5 45 5.7 -5.7 -5.3 -5.2 5.9

FIG. 3. Dependence of chemical shifts of resonances in Fig. 2 upon pH* for binary complexes of E. coli MB 1428 dihydrofolate reductase with methotrexate, aminopterin, folate, methopterin, and trimethoprim. - - - deuterium-exchangeable resonances; 0, resonances corresponding to more than 1 proton; p, standard histidine titration curves, except for Curve 4 of the methotrexate complex.

TABLE II

Chemical shifts of acid orprotonated forms of histidine residues of E. coli dihvdrofolate reductase

Ligand Histi- dine

Assign- ment Metho- Aminop-

trexate terin Folate Methop- m;;;o. twin Drim

1 124 2605 2596 2580 2580 2605 2 141 2664 2650 2652 2635 2648 3 149 2637 2642 2640 2610 2626 4 114 2608 2606 2640 -2610 2600 5 45 2646 -2640 -2650 -2650 -2530

cantly upfield 6(A) (chemical shift of the neutral form) compared to the other four complexes. Histidines 4 and 5 showed the most variability between the complexes. Histidine 4 was virtually identical for the methotrexate and aminopterin complexes as was histidine 5. The methopterin complex had lower pK’ values by about 0.25 and 0.45 units for 4 and 5, respectively, but had about the same chemical shifts for 4 and 5 as the methotrexate complex. Histidine 5 was about the same in chemical shift and pK’ in the methopterin and folate complexes. Histidine 4 in the folate complex was different in both pK’ and S(HA’) from the other three folates. A comparison of the trimethoprim and methotrexate complexes showed the same pK’ and the same 6(HA’) within +16 Hz for histidines 1 to 4, but histidine 5 had a significantly more upfield 6(HA’) for the trimethoprim complex. The correspondences in

6(HA’) and pK’ for the titrating histidines were the basis for the suggested assignments in Table I for complexes other than the methotrexate complex. This assumed that the local environment of a particular histidine was about the same in each of the five complexes for at least 4 of the 5 histidines.

Shown in Fig. 4 are the aromatic regions of the 300 MHz ‘H nmr spectra of the binary methotrexate complexes of E. cob MB 1428 dihydrofolate reductase and of DTNB- and NBrSuc- modified enzyme. The labeled resonances corresponded to histidine residues numbered as in Figs. 5 and 6. Between 1900 and 2130 Hz the three spectra were similar; this was the region of the ‘H nmr spectrum where the 3’,5’-protons of the 4 tyrosine residues of the reductase have their resonances (21). Also, as discussed below, the ‘H nmr characteristics of 4 of the 5 histidine C-2 protons of the three enzyme forms were similar. Thus, insofar as the ‘H nmr characteristics of the


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His Titrations of Dihydrofolate keductase 8147

NBS

I I, 1 I I, I , I, I

.x300 2400 2000

I 1 1 I 1 I I I I I I IO 9 6 7 6 wm

FIG. 4. Aromatic region of the 300-MHz ‘H nmr spectra of the binary methotrexate (MTX) complexes of E. coli dihydrofolate reductase and of the enzyme specifically modified with N-bromosuccinimide (NBS) and DTNB. The DTNB-modified enzyme had 1 cysteine modified, Cys 152, and the NBrSuc-modified enzyme had 1 hi&dine modified. The top, middle, and bottom spectra were at pH* 7.61, 7.60, and 7.37, respectively, and at 25°C.

tyrosine and histidine resonances reflect the conformation of the protein, the nmr spectra suggested that the DTNB- and the NBrSuc-modified enzymes had similar conformations to the unmodified enzyme. The differences between the three spectra in the 2130- and 2230-Hz region may be in part attributed, in the DTNB-modified enzyme, to resonances of the protons of the thionitrobenzoate moiety on Cys 152, and in the NBrSuc-modified enzyme to the small proportion of Trp residues that were also modified by NBrSuc when the reactive histidine is modified (5).

The direct comparison of the ‘H nmr histidine titrations of the binary methotrexate complex of E. coli dihydrofolate reductase and DTNB-modified enzyme shown in Fig. 5 and numerically summarized in Table III gave the following results. First, 4 of the 5 histidines had the same pK’. The pK’ of histidine 2 increased 0.2 unit in the modified enzyme, possibly a significant increase. Second, 3 of the 5 histidines titration data could be fitted to standard titration curves with the same chemical shift at acid pH 6(HA+) in both DTNB-modified and unmodified enzyme. Histidine 3 had 6(HA+) 19 Hz to

270C

260C

s HZ

250C

240C

23OC

I-

,-

,-

)-

I-

I

-0 E(l428) MTX

--A DTNB - Modified

1

6 7

PH*

8

FIG. 5. Direct comparison of the dependence of chemical shifts upon pH* for the binary methotrexate (MTX) complex of E. coli dihydrofolate reductase and of DTNB-modified E. coli dihydrofolate reductase. U, unmodified enzyme; A- - -a, the DTNB-modified enzyme. Data points corresponding to exchangeable resonances were omitted (Fig. 3). The sigmoidal carves are the standard histidine titration curves that gave the best visual fit to the data points; the pK’ values and extrapolated chemical shifts at pH extremes are given in Table III.

lower field, a possibly significant change, and histidine 4 was 50 Hz to lower field in the modified enzyme. Finally, 4 of the 5 histidines had the same 6 at alkaline pH in both enzyme forms. Histidine 2 was 61 Hz to lower field in the modified enzyme. Thus, DTNB modification of Cys 152 leads to a measurable alteration in the environments of 3 histidines. The negatively charged thionitrobenzoate moiety perturbs the environment of the acid or protonated form of histidine 4 and, to a lesser extent, that of histidine 3. The moiety perturbs the alkaline or uncharged form of histidine 2.

When E. coli MB 1428 dihydrofolate reductase was titrated carefully with N-bromosuccinimide to a 40% loss of enzyme activity as described by Williams (5), 1 histidine residue was modified and methionine and cysteine were partially oxidized. By comparison of the data summarized in Fig. 6 and Table III with that in Figs. 3 and 5 and Table III, it was clear that the affected histidine was histidine 5. The atypical reactivity of histidine 5 with NBrSuc may have been due to its unusually low pK’. In agreement with the data of Williams (5), the 40% inactivated enzyme appeared to have about the same conformation as the native enzyme. The high field resonances above 0.9 ppm or 270 Hz exhibited the same chemical shifts and intensities in the ‘H nmr spectrum of the binary methotrexate complex of the NBrSuc-modified enzyme as in the unmodified enzyme, which suggested that the tertiary structure near the side chains of aromatic residues of the enzyme were the same in modified and unmodified enzyme.

The histidine titration parameters for the histidine C-2 protons of the binary methotrexate complex of the NBrSuc- modified reductase and of the normal enzyme were roughly the same for histidines 1 and 3 (see Table III). Histidine 2 and


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4 in the NBrSuc-modified enzyme were more similar to the DTNB-modified enzyme than to the normal enzyme, which was probably due to the oxidation of Cys 152 to a negatively charged species by the NBrSuc. Williams (5) earlier noted the oxidation of the easily accessible cysteine, later identified as Cys 152 by Williams and Bennett (13), in the 40% NBrSuc- inactivated enzyme. The DTNB-modification of Cys 152 also introduced a negative charge at this site. Two interesting phenomena in the NBrSuc-modified enzyme are the doubling of histidine resonance 4 above pH* 7 and the low field position of the C-4 resonance of histidine 4. The doubling could be due to a conformational equilibrium similar to that noted by Meadows and co-workers (22, 23) in their studies of the histidine titrations of pancreatic ribonuclease, such as the two conformations for methotrexate found by Matthews et al. (3) in the 2 molecules of the asymmetric unit of the binary E. coli dihydrofolate reductase. methotrexate crystal.

There was a fifth titrating histidine in the NBrSuc-modified

27OC

260(

8 Hz

25Ot

2401

2301

1 I I

NBS - E(l428) : MTX

1 , \, I I I

6 7 6

PH”

FIG. 6. Dependence of chemical shift upon pH* for the hi- nary methotrexate (MZ’X) complex of NBrSuc (NBS)-modified E. di dihydrofolate reductase. Symbols as in Fig. 3.

enzyme whose titration parameters were close to those of normal histidine (15,19). However, this resonance had an area corresponding to only about 0.3 protons when compared to histidine 3. The area of this new resonance did not decrease noticeably during the pH* titration, suggesting that the miss- ing area was not attributable to exchange for deuterium in the medium, It was unclear what the chemical structure (or structures) of NBrSuc-modified histidine 5 was; NBrSuc modification of histidine can give rise to several products (24,25). At least 30% of NBrSuc-modified histidine 5 must have been aromatic like normal histidine and unmodified at position C- 2.

The two ‘H nmr spectra replotted for Fig. 7 were of the aromatic proton resonances of the binary methotrexate complex at 0.76 IIIM and pH* 7.27, before and during reaction with the histidine-specific reagent, ethoxyformic anhydride. The binary enzyme complex was incubated 12 min at 25°C with 0.05% ethoxyformic anhydride, and it took 15 min to obtain the spectrum. The resonances corresponding to most aromatic protons of the protein were unchanged during this reaction, suggesting that the overall structure of the protein was not much changed by ethoxyformylation of the histidines. Histi- dines 3 and 5 reacted completely during the experiment, while histidines 2 and 4 reacted partially, and histidine 1 reacted hardly at all. Histidines 3 and 5 probably reacted rapidly because of the favorable pK’, i.e. they were mostly in the uncharged form at pH 7.2, while histidine 1 was mostly in the positively charged ionic form and thereby reacted slowly. Histidine 2 probably reacted at moderate speed because of its intermediate pK’. Histidine 4 did not react rapidly despite its low pK’, possibly due to inaccessibility. Muhlrad et al. (26) and Melchior and Fahrney (27) have shown that ethoxyformylation shifts the pK’ of histidine from approximately 7 to

r

31 I 1 / I1 I I1 1, 1

0 2600 2200 1800 1400

HZ

FIG. 7. Low field portion of the ‘H nmr spectrum of enzyme methotrexate (MTX) before and during reaction with the histidine-specific reagent, ethoxyformic anhydride (EFA). Enzyme is 0.76 rnM at pH* 7.27 and 25’C in 0.50 ml; ethoxyformic anhydride was added (5 pl of 5% solution in CLHCLHzOLH) and reacted for 12

min at 25°C.

TABLE III

Comparison of histidine titrations of the binary methotrexate complexes of normal, NBrSuc-modified and DTNB-modified E. coli dihydrofolate reductase

E.methotrexate DTNB.E.methotrexate NBrSuc . E . methotrexate Histidine Assignment

PK’ [6(HA+)I [S(A)] PK’ lS(HA+)I l&A)1 PK’ 6(HA+) 6(A)

HZ HZ HZ HZ HZ HZ 1 124 8.0 2605 2305 8.0 2610 2310 8.0 2612 2312 2 141 7.2 2664 2293 7.4 2654 2354 7.5 2656 2318 3 149 6.6 2637 2337 6.7 2657 2346 6.9 2624 2324 4 114 6.2 2608 2308 6.2 2658 2310 6.2 2642 2342 & 2310 5 45 5.7 2646 2346 5.7 2646 2346 7.2 2652 2352


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4. Ethoxyformylation of E. coli dihydrofolate reductase. methotrexate led to enhancement of the intensity of the resonance at 2280 Hz (7.60 ppm); this resonance possibly corresponded to the C-2 resonance of the ethoxyformylated histidines, which would be in their neutral form at pH* 7.2 if they had pK’ values near 4.

DISCUSSION

Nuclear magnetic resonance studies on dihydrofolate reductase (14,2&J-40) have provided detailed information about specific sites on the enzyme, and the conformation of ligands on the enzyme. Among the more important residues in E. coli MB 1428 dihydrofolate reductase are the 5 histidine residues, which are at positions 45,114,124,141, and 149 in the sequence (3, 41, 42). These residues are important in maintaining the active configuration of the enzyme (5, 6). Each of the 5 residues is significantly different from the other 4 in its pK’, chemical shift, or ligand dependence as measured by ‘H nmr spectroscopy. In the following, we suggest assignments of specific histidine C-2 proton resonances to specific histidine residues on the basis of the histidine environments in crystalline enzyme. methotrexate complex. This of course assumes a high correlation between histidine environments in the crystal and in solution. The internal consistency of the assignments in explaining chemical shift and pK’ data, the consonance of the assignments with the results on different ligands, and the agreement of the results on chemically modified forms of the enzyme with these assignments suggest that this assumption is correct. Furthermore, the data suggests that the local environment of most of the histidines is not much affected by the different ligands in the methotrexate binding site, despite the fact that methotrexate and aminopterin are probably bound with their pteridine rings turned over relative to the conformation of folate and methopterin (4, 43).

Histidine C-2 resonance number 5 exhibits an approximately normal chemical shift in its neutral or unprotonated form but has quite low pK’ values for its complexes with folates. This resonance is assigned to histidine 45, whose local environment in its crystalline binary complex with methotrexate is shown in stereo view in Fig. 8. The side chain of histidine 45 is buried in a hydrophobic environment except for Ne2 which points into solvent. No negatively charged residues are nearby; the guanido groups of Arg 44 and Arg 98 are the closest charged groups and are about 11 A away. The low pK’ of histidine 45 is readily explained since protonation of the imidazole side chain requires breaking the hydrogen bond with Thr 46 and furthermore, NSl resides in a hydrophobic

environment that will stabilize the unprotonated form of the ring, As shown in a forthcoming article’ histidine 5, the histidine modified by NBrSuc, exhibits a dramatic change in its titration parameters in the ternary complex. For example its pK’ increases by +l.lO units in the ternary enzyme.methotrexate e NADPH complex. Matthews et al. (4) have shown that in the ternary complex of L. casei dihydrofolate reductase, which is closely related in three-dimensional structure to the binary methotrexate e E. coli reductase, the residue (Arg 44) which corresponds to His 45 in the E. coli sequence must have its side chain conformation completely readjusted to accommodate the pyrophosphate moiety of NADPH. The proximal negative charges on the pyrophosphate and the disruption of the hydrogen bond to Thr 46 would account for the pK’ rise in the ternary complex of histidine 5 of the E. coli enzyme, provided NADPH is bound the same way in the ternary complexes of the L. casei and E. coli enzymes. The normal chemical shift of the neutral form of the histidine is explainable by the absence of nearby aromatic groups; the nearest aromatic is Trp 47 which is about 11 A away. The great difference in the nmr titration parameters of histidine 5 in the binary trimethoprim complex when compared to the other folate complexes is possibly attributable to the fact that His 45 is the histidine in the enzyme*methotrexate crystal structure that is closest to the p-aminobenzoyl binding site, a site that trimethoprim cannot fill.

Histidine C-2 resonance 4 is the histidine which exhibits the most varied values of pK’ and chemical shift for the five complexes in Tables I and II; its pK’ ranges from 5.7 to 6.3. It is upfield shifted compared to histidine 3 by about 100 Hz or 0.33 ppm in enzyme. folate and by about 30 Hz or 0.1 ppm in the other complexes. It is suggested that this resonance corresponds to histidine 114, whose local environment is shown in Fig. 9. This histidine is part of p sheet F, and is above and between the peptide backbones in p sheets F and H and is well solvated. The side chain of His 114 is so far removed from the pteridine binding site that it is unlikely any charge here would contribute to chemical shift differences.

Tyr 128 hydrogen bonds with a ring nitrogen of histidine 114. Interestingly, another side chain (Lys 154) is in close proximity to the imidazole ring but has different conformations in each of the two crystallographically independent molecules. As a result, the e-amino nitrogen which resides only 5.5 A from the center of the imidazole ring in one molecule, is some 2 A further from this side chain in the second molecule. This suggests that there may be several conformational states of similar energy for at least one side chain in the vicinity of histidine 114. Recall that in the

FIG. 8. Stereo picture of the local environment of histidine 45 in crystal structure of binary methotrexate complex of E. coli MB 1428 dihydrofolate reductase (3). Shown are the non-hydrogen atoms of Ile 41, Met 42, Gly 43, Arg 44, His 45, Thr 46, Leu 62, Ser 63, Ile 94, Gly 95, Gly 96, Gly 97, Arg 98, and Val99. Oxygen and nitrogen atoms are indicated by blackening and shading, respectively.


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NBrSuc-modified enzyme resonance 4, which we assign to histidine 114, is a doublet about pH 7. The above discussion suggests that the splitting could arise as a consequence of a conformational equilibrium. The positive charge on Lys 154 and the hydrogen bond between the hydroxyl group on Tyr 128 and the unprotonated ring nitrogen of His 114 would account for the low pK’ of this histidine residue.

when both are fully protonated. This is proposed to be histidine 141, whose local crystalline environment is shown in Fig. 10. The histidine residue is a part of p sheet G, is between p sheets G and H, but is about half-exposed to solvent. The carboxyl group of Glu 139 is quite close to the ring of His 141;

Greenfield (6) has shown that several lysine residues are modified by ethoxyformic anhydride. Modification of Lys 154 probably would reduce the accessibility of His 114 to the solvent and thereby account for the reduced reactivity of histidine 4.with ethoxyformic anhydride. The hydroxyl group of Thr 113 is directly hydrogen-bonded to the 2-amino group of methotrexate. The hydrogen bond may be different for different ligands in the folate site and could thereby affect the position of histidine 114. It is also possible that Tyr 128 may occupy different positions, depending on which substrate or inhibitor is bound at the active site. Tyr 128 is part of a flexible loop which undergoes a conformational change when NADPH binds to the L. casei enzyme - methotrexate complex (4, 43). I f conformational changes in this loop can also occur as a result of binding substrates and inhibitors this could account for the sensitivity of the nmr parameters of histidine 4 to different ligands. Because of the close proximity of the Tyr 128 side chain to the imidazole ring of His 114, it is quite possible that small conformational changes that might occur upon binding folate could partially stack the two rings and, thus, the observed upfield shift of 0.33 ppm could be explained by a ring current mechanism.

Histidine C-2 resonance 2 has a moderately high pK’ (7.2 to 7.4) and has about the same chemical shift as resonance 3

it is 5.0 and 3.9 A from the two oxygens to the ring center. This proximal negative charge would make it easier to proton- ate His 141 and thereby give it a moderately high pK’. The center of the aromatic rings of Tyr 151, Phe 153, and Phe 140 are 4.7, 5.3, and 7.4 A from the histidine ring center, respectively, and could lead to the downfield shift of the nmr resonance of the histidine C-2.

The 2 His residues in the crystal structure of the binary methotrexate complex of E. coli dihydrofolate reductase (3) whose side chains are closest to Cys 152 are His 114 and His 141, assigned above as histidines 4 and 2, respectively. The centers of their rings are 6.6 and 8.1 A from the cysteinyl sulfur, respectively. The neutral form of histidine 2 and the protonated form of histidine 4 are the species most strongly perturbed by DTNB-modification. The neutral or uncharged form of his 2 is shifted +0.20 ppm downfield upon DTNB- modification of the enzyme, and the protonated form of his 4 is shifted +0.17 ppm downfield; these are the largest changes in histidine titration parameters for the DTNB-modified enzyme. The fact that different ionic species of the 2 histidines are perturbed by DTNB-modification suggests that the local environment of the 2 histidines have ionic characteristics different from one another. As noted above, His 114 and His 141 have a different ionic environment in the enzyme’s crystal structure. His 114 is near the positive charge on the e-amino group of Lys 154, while His 141 is near the negative charge on

ASP Ii6 0% 116

FIG. 9. Local environment of histidine 114. Atoms of Ala 7, Leu 8, Ala 9, Val 10, Asp 11, Leu 112, Thr 113, His Cys 152, and Lys 154 are shown. 151,

PHt 153

114, Ile 115, Asp 116, Tyr

PHE IS3

TYR

SER 150

ASN 1112

TIR 151

FIG. 10. Local environment of histidine 141. Atoms of Glu 139, Phe 140, His 141, Tyr 151, Cys 152, and Phe 153 are shown. The sulfur atom in Cys 152 is cross-hatched.


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the carboxyl group of Glu 139. The thionitrobenzoate moiety on DTNB-modified cysteine has a negative charge on its aromatic carboxylate group; this charge gives it the ability to make ionic interactions. The precise details of the interaction of the thionitrobenzoate moiety on DTNB-modified Cys 152 with His 114 and His 141 are not known. A possible mechanism for the moiety to interact more strongly with the appropriate ionic forms of histidine 2 and 4 goes as follows. The negatively charged carboxylate of DTNB-Cys 152 could interact in a direct ionic manner more strongly with the protonated form of His 114 (histidine 4) than the neutral form. This direct interaction of charged species would have to be fairly weak since the pK’ of histidine 4 is not significantly changed upon DTNB modification. On the other hand, the protonated form His 141 (histidine 2) which interacts strongly with the nearby side chain of Glu 139 could be free to interact with the aromatic ring of the DTNB in its neutral form. It should be noted, however, that a slightly different and equally plausible mechanism of DTNB-Cys 152 interaction with His 114 and His 141 would be consistent with stronger interaction of the DTNB moiety with the opposite ionic forms of His 114 and His 141. Thus, it is not possible to make an assignment of histidines 2 and 4 to His 114 and His 141, respectively, on the basis of the titration characteristics of the DTNB-modified enzyme alone. The small effects of DTNB modification upon histidine 3, assigned below as His 149, are possibly due to a small conformational change in /3 sheet /3H to accommodate the thionitrobenzoate moiety on Cys 152; /3H contains both Cys 152 and His 149.

Histidine C-2 resonance 1 has a relatively high pK’ (7.9 to 8.2) and is roughly 45 to 60 Hz upfield of resonance 3 when both are equally protonated. This is suggested to be histidine 124, whose local environment in the enzyme.methotrexate crystal is shown in Fig. 11. This histidine residue is part of a long loop at the enzyme’s surface connecting /3 sheets F and G, and is mostly exposed to solvent. The side chain of this

histidine residue is at the surface of the molecule and quite remote from the site of NBrSuc modification (His 45) and DTNB modification (Cys 152) as well as from the methotrexate and pyridine nucleotide binding sites. The nearest aromatic ring (on Phe 125) is centered 10.3 A away from the histidine ring center. There are 3 nearby charged residues. The distance from the histidine ring center to the carboxylate oxygen atoms of Asp 11 is 4.3 and 5.9 A and that of Asp 122 is 4.9 and 6.9 A, while it is 6.5, 7.5, and 8.1 A to the 3 guanido nitrogen atoms of Arg 12. The nearby charged residues, which are not quite as close as Glu 139 to His 141 but include more nearby negatively charged species, probably account for the pK’ near 8.

Histidine C-2 resonance 3 has a normal pK’ and a normal chemical shift and chemical shift change upon protonation (15). Depicted in Fig. 12 is the local environment of histidine 149, which is the /3 sheet F at the surface of the enzyme. Approximately seven-eighths of the solid angle about the residue is unobstructed so that it is essentially in the medium. There are no charged groups within 7 A of the ring center, and the nearest aromatic residue Trp 22 has its six-membered ring center 6.6 A away and its five-membered ring 5.9 A away. This histidine’s environment would not change much were the protein to be converted to an extended random coil It is proposed that histidine 149 is resonance 3, and that its prop- erties are those of a “normal” histidine under the conditions of measurement.

The dramatic change in the titration parameters of His 45 upon NBrSuc modification are doubtless due to the fact that this is the residue directly modified. The changes in the titration parameters of His 114 and 141 upon NBrSuc modification are similar to the changes noted upon DTNB modification of Cys 152. Oxidation of the cysteinyl sulfur of Cys 152 to an acid by NBrSuc would lead to the introduction of a negative charge at Cys 152, just as the DTNB modification leads to a negative change there. If the cysteinyl sulfur were

FIG. 11. Local environment of histidine 124. Atoms of Asp 11, Arg 12, Glu 120, Gly 121, Asp 122, Thr 123, and His 124 are shown.

FIG. 12. Local environment of histidine 149. Residues portrayed are: Pro 21, Trp 22, Asn 23, Leu 24, Ile 115, Asp 116, Ala 117, Glu 118, Val 119, Asn 147, Ser 148, His 149, and Ser 150.


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oxidized to a mixture of acids, this could possibly account for the doubling of histidine 4 in the NBrSuc-modified enzyme, although other mechanisms are also possible such as a conformational equilibrium based on the differences noted above for the environment of the histidine 114 side chain in each of the two crystallographically independent reductase molecules.

We have been unable to provide direct chemical evidence in support of the assignments made above. Cyanogen bromide peptides of the NBrSuc-modified enzyme were prepared according to Bennett et al. (42) but amino acid analyses of the peptides were the same as for the corresponding peptides of the native enzyme. The histidine exchange methods as described by Markley (44) were not suitable due to a lack of sufficient difference in the exchange rates of the 5 histidines.

The pK’ and chemical shifts of E. coli MB 1428 dihydrofolate reductase histidine residues reported here are the same within experimental error as earlier studies reported on methotrexate and folate complexes in 0.10 M NaCl, 0.005 M bis-Tris (14), despite the use of calcium borate buffer in the present data. The one significant difference is in histidine 4 in enzyme. folate, which exhibits a chemical shift 0.33 ppm upfield and a pK’ 1.2 units lower.

Recently, Birdsall et al. (28) have reported the histidine titration curves of L. casei dihydrofolate reductase, an enzyme whose amino acid sequence shows considerable homology to the E. coli reductase in the alignment proposed by Freisheim et al. (45). The L. casei enzyme does not have a histidine among its 6 histidines which has a low pK like histidine 5 in the E. coli enzyme. However, E. coli reductase histidines 1,2, 3, and 4 do have counterparts in the L. casei enzyme, including a histidine labeled HE: in the L. casei enzyme which is significantly different in folate and methotrexate complexes.

Achnowledgments-Thanks are due to J. K. Wu for expert tech- nical assistance and Dr. Graham M. Smith for help in figure preparation, both of Merck & Co., Inc.

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M Poe, K Hoogsteen and D A Matthewsbasis of the crystal structure with methotrexate and on chemical modifications.

Assignment of histidine C-2 protons in binary complexes with folates on the Proton magnetic resonance studies on Escherichia coli dihydrofolate reductase.

1979, 254:8143-8152.J. Biol. Chem.

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