Crystal Structures of Escherichia coli and Lactobacillus

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 22, Issue of November 25, pp. 13650-13662, 1982 Printed in U.S.A.

Crystal Structures of Escherichia coli and Lactobacillus Dihydrofolate Reductase Refined at 1.7 A Resolution I. GENERAL FEATURES AND BINDING OF METHOTREXATE*

(Received for publication, June I, 1982)

Jeffrey T. BolinS, David J. Filman, David A. Matthews, Ronald C. Hamlinfj, and Joseph Kraut From the Departments of Chemistry and $Physics, University of California, San Diego, La Jolla, California 92093

X-ray data have been extended to 1.7 A for a binary complex of Escherichia coli dihydrofolate reductase with methotrexate and a ternary complex of Lactoba- cillus casei dihydrofolate reductase with methotrexate and NADPH. Models for both structures have been refined to R factors of 0.15 and include parameters for fixed and liquid solvent.

“he two species of dihydrofolate reductase resemble one another even more closely than was thought to be the case prior to refinement. Several new structural features have also been discovered. Among them are a cis peptide linking Gly-97 and Gly-98 (L. casei numbering) in both species, an (Y helix involving residues 43 through 50 in the E. coli enzyme, and the existence of what may be a specific hydration site on exposed (Y

helices. Refinement has led to a revised description of the

details of methotrexate binding. We now see that a fixed water molecule mediates the interaction between methotrexate’s 2-amino group and Thr-116 (L. casei numbering) and that the inhibitor’s 4-amino group makes two hydrogen bonds with the enzyme (instead of one). Other revisions are also discussed.

A hypothetical model for substrate binding is proposed in which the pteridine ring is turned upside down while all protein and solvent atoms remain fixed. Asp- 26 in this model is hydrogen bonded to the substrate’s 2-amino group and to N3.

Earlier publications from this laboratory presented initial reports on the x-ray crystallographic structures of two bacterial dihydrofolate reductases, one a binary complex of the Escherichia coli enzyme with the inhibitor methotrexate (Matthews et al., 1977) and the other a ternary complex of the Lactobacillus casei enzyme with both methotrexate and the cofactor NADPH (Matthews et al., 1978 and 1979). Re- cently we extended this series to include a representative vertebrate dihydrofolate reductase, namely the chicken enzyme in a ternary complex with a phenyltriazine inhibitor and NADPH (Volz et al., 1982). The nominal resolution of the x- ray data in those studies was 2.5 A for both bacterial enzymes and 2.9 A for the chicken enzyme.

The structural models described in our earlier reports were based on interpretation of initial electron density maps com-

* This work was supported by Research Grants CA 17374, GM 10928, and RR 00757 from the National Institutes of Health. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Department of Biological Sciences, Purdue Uni- versity, West Lafayette, IN 47907.

puted from isomorphous replacement-derived phases, and because they had not been subjected to exhaustive crystallographic refinement, the models were necessarily somewhat preliminary in nature. They nevertheless provided a great deal of information concerning backbone chain folding in the dihydrofolate reductase molecule and furnished the founda- tion for a comparative study of species-to-species variation in the structure of the enzyme. Additionally, these initial structures provided valuable preliminary information about the geometry of inhibitor and cofactor binding.

But much more can be extracted from the x-ray data than is evident in the isomorphous replacement-phased electron density maps. Several years of experience in a number of protein crystallography laboratories, our own included, has dramatically demonstrated that early models of this sort are often unreliable in important details and that much more precise and detailed structural information can be derived by crystallographic refinement. We therefore decided to make the quite substantial investment of effort required to extend the resolution of the data to the accessible limit, about 1.7 A, and to refine the models as far as possible. In this and the immediately following communication (Filman et al., 1982) we briefly describe the high resolution refinement and, in considerably more detail, our refined models for the E. coli and L. casei dihydrofolate reductase structures. The two bacterial dihydrofolate reductases are considered together throughout. A similar refinement of the chicken enzyme structure is currently in progress.

This paper focuses on overall conformation and features of secondary structure, the role played by the hundreds of fixed solvent molecules that are part of the crystal structures, binding of the methotrexate inhibitor molecule to the enzyme, and a hypothetical model for substrate binding. The immediately following paper describes the stereochemistry of cofactor binding and discusses some implications of what is now known about both substrate and cofactor binding geometry for the enzymic mechanism of dihydrofolate reductase.

We have attempted throughout both papers to call atten- tion to those instances where significant revisions have had to be made to our earlier reports as a result of crystallographic refinement.

EXPERIMENTAL DETAILS AND STRUCTURE REFINEMENT

E. coli dihydrofolate reductase was a gift from M. Poe (Merck Institute for Therapeutic Research). It had been pu- rified by methotrexate-agarose affinity chromatography from a cell culture of strain MB1428 (Poe et al., 1972) and supplied to us as a 1:1 binary complex with methotrexate. Crystals of the binary complex, grown from 20% ethanol buffered at pH 6.8 with 0.01 M histidine hydrochloride containing 5 mM calcium acetate, have space group P6,, with unit cell param-

13650

Crystal Structures of Bacterial Dihydrofolate Reductases 13651

eters a = b = 93.22 A, and c = 73.56 A. The asymmetric unit contains two molecules. For x-ray data collection the mother liquor was replaced by buffered 35% ethanol.

L. casei dihydrofolate reductase, supplied by R. Kisliuk, had been isolated from a dichloromethotrexate-resistant strain (Crusberg et al., 1970). It was further purXed by E. Pastore on a pteroyllysine-agarose affinity column (Pastore et al., 1974) and crystallized by us as the ternary complex, with equimolar methotrexate and NADPH, out of 55% saturated ammonium sulfate buffered at pH 7.4 with 0.01 M Tris. Crys- tals of the ternary complex also had space group P61 (presumably a coincidence), but with unit cell parameters a = b = 71.86 A, c = 93.38 A and only one molecule per asymmetric unit. For x-ray data collection the mother liquor was replaced by buffered 85% saturated ammonium sulfate.

For both kinds of crystals most of the x-ray diffraction data to a Bragg spacing of 1.7 A were collected on the multiwire area detector diffractometer of Xuong and coworkers (Cork et al., 1974; Xuong et al., 1978). Additional low angle data were measured on a Hilger-Watts diffractometer. The L. casei data set, which is 92% complete to 1.7 A, consists of 24,380 unique reflections with a weighted internal R factor for replicated intensities of 0.06 (method of Monahan et al., 1967). The E. coli data set, which is 88% complete to 1.7 A, contains 32,554 unique reflection intensities with an internal R factor of 0.07. Data from four crystals were combined to form each set, yielding an average of five measurements for each unique reflection in both cases.

The starting point for refinement was, in both cases, an initial model derived from isomorphous replacement-phased electron density maps (Matthews et al., 1977, 1978). A 2257- atom subset of the E. coli model, containing 87% of the structure, was considered sufficiently reliable to be used in the fist F,. calculation and gave an R factor of 0.49 for 2.5 A data. The starting L. casei model contained 1379 atoms and accounted for all of the structure, although the portions corresponding to residues 87-92 and 133-136 were rather specula- tive because of poor definition in the MIR-phased map. An initial F,. calculation gave an R factor of 0.45 at 2.43 A.

The general strategy of refinement was repeatedly to re- build doubtful parts of the model with the aid of 12F0 - F,I, and IF,, - F,I maps computed with F, phases, alternating rebuilding with gradient/curvature or Hendrickson and Kon- nert (1980) least squares optimization of positional and thermal parameters. In the early stages only a single overall temperature factor was refined, but later individual isotropic temperature factors were introduced. Rebuilding was carried out on an Evans and Sutherland Picture System I using locally developed software written by Stephen A. Dempsey.’ In the later stages fixed solvent molecules were added, assigned individual occupancy factors, and refined along with the rest of the structure. Only solvent sites of one-third or greater occupancy were retained.

Particularly in the case of the L. casei structure, refinement progressed at an exceedingly slow pace initially, reaching an R factor of 0.31 after 216 cycles of model reconstruction, with 20% of the protein still represented by uninterpretable electron density or no electron density at all. In retrospect, we believe that slow convergence was probably due to failure of conventional structure factor calculations to allow for the contribution to low angle scattering by liquid solvent within the unit cell. The latter makes up 60% of the unit cell volume in the L. casei dihydrofolate reductase crystals, as compared with a solvent content of about 43% for other more typical protein crystals (Matthews, 1968).

’ S. A. Dempsey, unpublished programs.

The liquid solvent problem was finally solved by explicitly incorporating the solvent region into F, calculations by a method similar to one tested by Stroud and coworkers in the refinement of trypsin.’ In this method a uniform electron density distribution is fist erected throughout the asymmetric unit on a finely spaced grid. The spacing between grid points was chosen to be 0.75 A, or less than half the wavelength of the highest order Fourier term, and the electron density of each grid point was initially set equal to the electron density of liquid water, 0.34 e/A“. To establish the solvent boundary we then identified all grid points within a fixed radius of any protein atom; such points were considered to be within the excluded volume and assigned zero solvent density. Exclusion radii ranging from 2.5 to 3.5 A were tested for minimization of R; (see below) in a series of F, calculations, and although a value of 2.65 A turned out to be optimum, the residual was in fact found to be relatively insensitive to the precise value of this parameter. The contribution by the liquid solvent to the total structure factor was then taken as the Fourier transform of the resulting electron density distribution, (the “solvent plateau”) multiplied in reciprocal space by a Gaussian smooth- ing function, exp[-C(~in8/h)~], and by a linear scale factor, K,, both again chosen by systematic trial and error to minimize R2’. The value of C varied little throughout the course of refinement, ranging from 30 to 50 A’. On the other hand K, was found to vary as a function of the completeness of the model, in the case of the L. casei refinement decreasing the effective solvent density from an initial value of 1.5 e/kR to a final value of 0.46 e/k’, which is only slightly greater than the expected value of 0.40 e/A” for 85% saturated ammonium sulfate (Bragg and Perutz, 1952). Despite their variability, the residual was not sensitive to the precise values of C and K.,, and we found it unnecessary to change these parameters with every cycle of refinement.

In later stages of refinement a computationally simpler but nearly as effective approach to modeling the liquid solvent was adopted. It is conceptually identical with one of several methods tested by Moews and Kretsinger (1975) in their refinement of parvalbumin, except that rather than using it simply as a final correction to F,, we applied it continuously, at each round of least squares and F,. calculations. In this approximation, the liquid solvent region is represented by what would be left after subtracting a suitably smeared-out protein molecule from a uniform field of electron density. More precisely, each structure factor, computed from the protein atoms and fixed solvent atoms, is multiplied by the expression

klexp[-BIs’] X (1 - k2exp[-Bzs’]) (1)

where s = sinB/X, k l and B1 are overall scale and temperature factor adjustments, k~ relates the electron density of the liquid solvent to that of the protein molecule, and BZ serves to smear out the protein shape. Optimal values for all four adjustable parameters were chosen by systematic trial and error to minimize Rz’. Again, the residual is fairly insensitive to the precise values of kz and BB, and they need not be redetermined unless the model has been significantly altered. Values for k2 ranged from 0.7 to 0.9, reflecting the slightly lower electron density of the solvent as compared to that of the protein. B2 ranged from 200 to 400 A2; these large values correlate with the observation that the effect of liquid solvent scattering is important only a t low resolution. Optimum values for k2 and B2 at the end of refinement, for example in the case of the E. coli structure, were 0.90 and 375 A’, in agreement with Moews and Kretsinger’s values of 0.87 and 311 A’. An advantage of

J. Chambers, personal communication.

13652 Crystal Structures of Bacterial Dihydrofolate Reductases

this procedure is that it can either be applied after a conventional F, calculation is completed, or it can be implemented during the calculation by modifying atomic scattering factors. We preferred the latter approach because, by using modified scattering factors within the Konnert-Hendrickson program, the solvent model was included in the calculation of the least squares derivative matrix and right-hand side vector, as well as in the F, calculation.

Throughout the foregoing we have referred to minimization of a residual R; by systematically varying certain adjustable parameters. The quantity RP' is a version of the more usual R2 in which the adjustable scale contant, k, in the expression ( 1 F, I -k IF, 1 )2 is replaced by its own least squares value. The resulting expression for the residual is then

At the end of the L. casei dihydrofolate reductase refinement, the Moews and Kretsinger liquid solvent model resulted in an R value only 0.005 greater than that achieved by refinement in the presence of the solvent plateau model. It is interesting and somewhat surprising that this more approximate method gives such good results since a real valued multiplicative factor obviously cannot itself alter calculated phases, whereas the more elaborate solvent plateau procedure changed calculated phases by an average of 10". Nevertheless, it is clear that the coefficients F, - Fc will contain large errors at low resolution unless F, includes the liquid solvent correction. The important point to be stressed here is that inclusion of an amorphous solvent model, by either method, in the structure factor calculations for the L. casei dihydrofolate reductase structure caused a dramatic improvement in the interpretability of difference maps at a point of premature convergence and permitted refinement to converge to a much lower R factor fairly rapidly. However, we did not notice a similar improvement in the interpretability of difference maps when a liquid solvent model was included in the refinement of the E. coli dihydrofolate reductase structure.

In the course of numerous cycles of model rebuilding, many isolated peaks of electron density were identified as fixed solvent molecules and included in subsequent structure factor calculations. However, only those solvent molecules were retained which continued to refine with an occupancy of 0.3 or greater and which continued to produce a significantly positive peak in difference maps when they were later deleted from Fc calculations. Ultimately the E. coli model contained 428 fixed waters bound to the two protein molecules of the asymmetric unit, two chloride ions (one in each pyrophos- phate-binding site), and one calcium ion. Identification of these ions was based on electron counts and chemical environments. The final L. cusei model included 264 fixed water molecules, but no bound ions were in evidence even though the medium contained 3.3 M ammonium sulfate. I t is unlikely that we would be able to distinguish between water molecules and ammonium ions, but sulfate ions should be identifiable at the resolution of this study.

The R factor for the E. coli model at the termination of refinement was 0.155, calculated over 32,554 unique reflections to 1.7 8. With the exception of Glu-130 and Asp-131, all side chains are accounted for in the final electron density map of one or both molecules in the asymmetric unit. The latter two residues, in both molecules, exhibit interpretable density only as far as Cy and CP, respectively. A single correction to the revised E. coli MB1428 sequence (Bennett et al., 1978) is suggested by our final model. In the published sequence residue 142 is listed as Asn; based on hydrogen bonding we believe it is likely to be an Asp. The final model for the protein

plus bound methotrexate composing a full asymmetric unit contains 2574 nonhydrogen atoms. A total of 28 protein atoms are not included, all of them in side chains.

In the case of the L. casei enzyme, a 1375 nonhydrogen atom model for the protein, NADPH and methotrexate ac- count for all 162 residues and give an R factor of 0.152, calculated over 24,380 unique reflections to 1.7 8. With the possible exception of Lys-51, the side chain of which is missing four atoms and which is either highly disordered or else may actually be an alanine, the primary sequence of the model agrees with that reported by Bitar et al. (1977) for dihydrofolate reductase isolated from an independently derived methotrexate-resistant strain.

These refinement statistics correspond to a mean uncer- tainty in atomic positions of about 0.15 8 (method of Luzzati, 1952). Bond lengths in both refined models differ from standard bond lengths by a root mean square deviation of 0.065 8, and the root mean square difference between individual isotropic temperature factors assigned to covalently linked atoms is about 3 1\' in the E. coli model and 4 A2 in the L. casei model. When the L. casei structure was subjected to further least squares refinement in the absence of geometrical restraints and refinement was allowed to proceed to apparent convergence, the root mean square shift in atomic coordinates was 0.1 a and bond lengths deviated from standard values by 0.1 A. The R factor decreased by only 0.01 as a result of this procedure.

As a measure of the extent to which refinement has altered the initial structures, it is useful to examine the resulting shifts in atomic positions. For the L. casei ternary complex the overall root mean square shift was 1.6 8, and for the E . coli binary complex the corresponding figure was 1.3 8. However, these rather large values reflect the influence of a few regions that were poorly defined in the original MIR phased maps and a perhaps more reliable indicator of the extent to which refinement has changed the initial models is the median overall shift in atomic position, which was 0.75 8 for both structures.

Coordinates for the refined models of the E. coli and the L. casei dihydrofolate reductase structures have been deposited with the Brookhaven Protein Data Bank (Bernstein et al., 1977).

GENERAL DESCRIPTION OF THE DIHYDROFOLATE REDUCTASE MOLECULE

Both E. coli and L. casei dihydrofolate reductase fold into an eight-stranded /3 sheet consisting of seven parallel strands and a single COOH-terminal antiparallel strand. Four a helices are inserted into the long loops of chain connecting successive /3 strands, and thus dihydrofolate reductase belongs to the a/fl class of proteins defined by Levitt and Chothia (1976). Figs. 1 and 2 provide an overall view of the backbone geometries of the E. coli binary complex and the L. casei ternary complex and depict the relationship of bound NADPH and methotrexate to the various elements of secondary structure. As can be seen from these figures, the two molecules have almost identical overall geometries. For a comparison of the dihydrofolate reductase folding pattern to that of the coenzyme binding domain of the NAD'-dependent dehydrogen- ases, see Matthews et al. (1978).

REVISIONS TO BACKBONE CONFORMATION

Refinement of E . coli and L. casei dihydrofolate reductase has yielded several significant revisions of our earlier picture of these structures. Some involve the substrate-binding site or the NADPH-binding site and permit us to see certain details more accurately and at greater resolution, with interesting

Crystal Structures of Bacteric

FIG. l. Backbone ribbon drawing of the E. coli dihydrofolate reductase enzyme-methotrexate binary complex. ,f3 strands (represented by arrows) and a helices are labeled. The approximate position of every tenth residue is indicated.

FIG. 2. Backbone ribbon drawing of the L. casei dihydrofolate reductase enzyme-methotrexate-NADPH ternary complex. ,f3 strands (represented by arrows) and a helices are labeled. The approximate position of every tenth residue is indicated.

implications for the enzyme mechanism. These will be discussed in subsequent sections and in the following paper. In this section we will concentrate on a number of newly discovered features of the backbone conformation.

Perhaps the most important such feature is the revised backbone geometry of residues 97-99,lc (94-96,ec).” The conformation of this chain segment is now identical in all three

:I The symbols “ec” and “IC” are appended to residue numbers to indicate the E. coli and L. casei enzymes, respectively; numbers relevant to only one of the two molecules in the E . coli reductase structure are distinguished by adding a “1” or “2”.

z l Dihydrofolate Reductases 13653

molecules (L. casei and both E. coli) and consists of a parallel p bulge involving Ala-97,lc and Gly-98,lc (Ile-94,ec and Gly- 95,ec), followed immediately by a cis peptide bond between Gly-98,lc and Gly-99,lc (Gly-95,ec and Gly-96,ec). Moreover, crystallographic refinement of the chicken liver dihydrofolate reductase structure, currently underway, indicates that there is a parallel ,B bulge followed by a cis Gly-Gly peptide bond a t the corresponding location in that molecule as welL4 In fact, alignment of all known dihydrofolate reductase sequences (Volz et al., 1982) suggest that a cis Gly-Gly peptide bond is a conserved feature of the enzyme structure, since all the sequences contain Gly-Gly at this point. Although cis peptide bonds not involving the sequence X-Pro are rare, they are not unknown. Three have been observed in carboxypeptidase A (Rees et al., 1981). It is interesting that in both dihydrofolate reductase and carboxypeptidase A these cis peptide bonds occur near the active site and at the carboxyl ends of /3 strands.

A second cis peptide bond has been found, but only in the L. casei structure, between Arg-52,lc and Pro-53,lc, and is one feature in a revised model for the backbone conformation of the loop including segment 50-55,lc.

As we will demonstrate later in this paper, both cis peptide- containing regions interact with bound methotrexate. Fur- thermore, in the L. casei ternary complex the revised conformation of residues 96-99,lc positions the carbonyl oxygen of Ala-97,lc near the nicotinamide ring of bound coenzyme. The mechanistic implications of this fact will be discussed in the following paper.

In the case of the refined L. casei structure, several peptide planes in the loop between PA and aB, residues 9-23,1c, are rotated by 90” or more from their orientation in our initial model. As a result, the interactions between this loop and the coenzyme, which will be discussed in the following paper, differ somewhat from those described earlier (Matthews et al., 1979). The analogous loop, 10-24,ec, of E. coli molecule 1 was poorly defined in the original electron density map and, on the basis of the refined structures, we now know that the two molecules of the E. coli binary complex differ by more between themselves in this loop than do E. coli molecule 2 and the L. casei ternary complex. In the following paper we will again refer to this point as it relates to the possibility of conformational changes being induced by coenzyme binding.

A slight change in the conformation of residues 45 and 46,ec in both E. coli molecules has established that residues 43-50,ec do, after all, form an a helix. This helix, aC, was not well defined in the original MIR-phased electron density map and was, therefore, not mentioned in our earlier publication (Matthews et al., 1977) concerning the E. coli enzyme, nor was it depicted in the accompanying figures. However, it corresponds exactly to the helix aC (residues 42-49,lc) found in the L. casei enzyme (Matthews et al., 1978).

A general result of refinement is that the backbone geometries of the two bacterial dihydrofolate reductase species have become even more alike. An initial root mean square difference of 1.7 A between 142 structurally equivalent a- carbon atom positions (Matthews et al., 1978) was reduced to 1.1 A, emphasizing once again the great similarity between the two molecules. Those minor differences that do occur are found mainly in loops connecting elements of secondary structure. These elements of secondary structure are essentially the same in the two species, homologous p strands or a helices differing from one another only in that they may be one or two residues longer or shorter (see below). Such close structural homology might seem remarkable, considering that a sequence alignment of dihydrofolate reductases from E. coli

D. d. Filman, work in progress.


and L. casei (Volz et al., 1982) yields only 27% identities. However, similarly rigid architectural conservatism coupled with a similar degree of sequence variability is also seen among the serine proteases (Jurasek et al., 1976; Brayer et al., 1978), the acid proteases (Tang et al., 1978), the globins (Takano, 1977), and the C-type cytochromes (Dickerson et el., 1976; Argos and Rossmann, 1979).

The two crystallographically independent E. coli molecules in the asymmetric unit are now almost identical stereochem- ically. They are related by a 2-fold rotation axis which is nearly perpendicular to both the a and c crystal axes, plus an insignificant translation of less than 0.2 A. When the two molecules are superimposed by this operation, the average difference between equivalent backbone atoms is 0.4 8, to be compared with 1.0 A when the initial unrefined models are similarly superimposed. Only two chain segments now differ by more than 1.0 A between the two molecules, namely residues 14-19,ec and 121-124,ec. If these 10 residues are removed from the comparison, the average difference is reduced to 0.3 A. One cause of the structural difference in this region is evidently the coordination of a hydrated Caa+ ion in a space between three molecules, to be described below.

SECONDARY STRUCTURE: f i SHEET

An eight-stranded ,8 sheet, encompasing 35% of the poly- peptide sequence, spans the center of the dihydrofolate reductase molecule. Residues participating in P sheet hydrogen bonds and composing individual strands in the two species are as follows: PA: 1-7,lc 1-8,ec; PB: 36-42,k 39-43,ec; PC: 56-63,1c, 58-63,ec; PD: 74-76,1c, 73-75,ec; BE: 93-98,1c, 89-95,ec; PF: 111-119,1c, 108-116,ec; PG: 135-144,1c, 132-141,ec; OH: 152-161,1c, 150-159,ec. These p strands are all parallel except for PH, which is antiparallel to the rest. The order of adjacent strands, proceeding from left to right in Figs. 1 and 2, is G-H-F-A-E-B-C-D.

As a consequence of refinement, we can now identify three disruptions in the P sheet hydrogen-bonding pattern that are common to the L. casei and both molecules of the E. coli dihydrofolate reductase structure. One of these is a “classic” /3 bulge as described by Richardson et al. (1978), involving Val-139 and Ser-140,lc (Val-136 and Phe-137,ec) at the NHZ- terminal end of PG and Val-157,lc (Ile-155,ec) in the antiparallel strand PH. It is curious that this P bulge becomes totally disrupted (a “P blowout”) in the chicken dihydrofolate reductase structure (Volz et al., 1982) by a six-amino acid insertion into PG. Another ,B bulge, but this one involving parallel strands, occurs just before the Gly-Gly cis peptide at the carboxyl end of BE. Here the backbone carbonyl of Ala-97,lc (Ile-94,ec) points out of the plane of the sheet into the substrate-binding site, and its role in the hydrogen-bonding pattern is assumed by the carbonyl oxygen of Gly-98,lc (Gly- 95,ec), which accepts a hydrogen bond from the peptide nitrogen of Gly-42,lc (Gly-43,ec). Finally, a second parallel P bulge occurs at the NH2-terminal end of PF, where the peptide nitrogen of Asp-ll1,lc (Gln-108,ec) hydrogen bonds to the carbonyl oxygen of Thr-1,lc (Ile-P,ec), replacing the peptide nitrogen of Thr-112,lc (Lys-109,ec).

Another somewhat unusual P structure occurs in the L. casei ternary complex and in molecule 2 of the E. coli binary complex, involving residues 11-14,lc (12-15,ec) and 125-128,lc (122-125,ec). Here, a short piece of antiparallel hydrogen- bonded structure which is independent of the main ,B sheet exhibits an unusual hydrogen-bonding pattern. It is included in Fig. 3 of the following paper where we depict the nicotinamide-binding site in detail. This feature may be described as a consecutive pair of classic P bulges, with the carbonyl oxygens of Gly-14,lc and Thr-126,lc accepting two hydrogen

bonds each from the peptide nitrogens of Asp-125,lc and Thr- 126,lc and Ile-13,lc and Gly-l4,lc, respectively. In the E . coli sequence the corresponding residues are Ile-14,ec; Gly-15,ec; Asp-122,ec; and Thr-123,ec. As a result the two strands are tightly linked by four hydrogen bonds over the length of two residues, instead of over three residues as in a normal antiparallel P sheet, and the side chains of Ile-l3,lc, Asp-l25,lc, and ThI”126,lc are all on the same side of the sheet. Since these side chains all participate in binding the nicotinamide nucleotide portion of NADPH (see the following paper) and are strictly conserved in all known dihydrofolate reductase sequences (except that Thr-126,lc is replaced by serine in the bovine liver sequence (Lai et al., 1982)) this latter character- istic must be functionally important.

Molecule 1 of E. coli structure deviates from the above pattern principally because the coordination of a hydrated Ca2+ ion by residues 17-18,ecl has pulled residues 14-19,ecl away from 121-124,ecl toward the carboxyl end of the helix aC. Among the adjustments to the backbone torsion angles that accompany this conformational change are differences between molecule 1 and molecule 2 of 180” and 90” in $14 and @I5, respectively. The possibility of a backbone hydrogen- bonding arrangement in molecule 1 like that described above for molecule 2 and the L. casei enzyme is thus eliminated.

As mentioned above, the Ca’+ ion occupies a space in the E. coli dihydrofolate reductase crystal between three protein molecules, namely molecuIes 1 and 2, and the third (2’) symmetry related to molecule 2 . Five water molecules (waters 490,642-644, and 660) and the carbonyl oxygen of Ser-135,ecZ’ are coordinated to the Ca2+ in octahedral geometry. Four of these water molecules are in turn hydrogen bonded to the polar side chains of Glu-l7,ecl, Asn-l8,ecl, Glu-134,ec2’, and Asp-l42,ec2 and to the carbonyl oxygen of Ala-l43,ec2 (see Fig. 3). Although Baccanari et al. (1975, 1981) have demonstrated cation inhibition for both isozymes from E . coli strain RT500, because of its intermolecular location we believe that the Ca” binding by the crystalline enzyme described above is a phenomenon of crystal packing and is probably not corre- lated with the results of those solution studies.

SECONDARY STRUCTURE: HELICES

On the basis of backbone torsion angles and hydrogen- bonding patterns we have identified four a helices in both the L. casei and the E. coli dihydrofolate reductase structures. Two of these, aB (residues 23-34,lc; 24-35,ec) and aC (residues 42-49,lc; 43-50,ec) are identical in the two species with respect to both the number of residues involved and their backbone hydrogen-bonding geometry. Residues 77-86,ec constitute the helix aE in the E . coli structure, but a two-residue insertion in the L. casei molecule adds an extra half-turn at the carboxyl end of the corresponding helix, residues 78-89,lc. The helix aF includes nine residues in both species (99-107,lc and 96- 104,ec), but there is a slight difference between the two in the arrangement of hydrogen bonds at the carboxyl terminus. In the L. casei molecule a F ends with a 310 hydrogen bond between the peptide nitrogen of Lys-107,lc and the carbonyl oxygen of Thr-l04,1c, whereas in the E. coli reductase all the hydrogen bonds are the standard 3.413 type and the helix is terminated by Pro-105,ec.

As has been noted for other refined protein structures, the backbone carbonyl groups in all the helices in both species of the dihydrofolate reductase molecule are splayed slightly out- ward, away from the helix axis (Baker, 1980 and references cited therein). A phenomenon which may be related, but which has not been commented upon previously as far as we are aware, is a distinct tendency for the hydrogen-bonded carbonyl oxygen atoms on the solvent exposed side of a helix

Crystal Structures of Bacterial Dihydrofolate Reductases 13655 n

FIG. 3. The intermolecular calcium ion-binding site in crystalline E. coli dihydrofolate reductase. Portions of three protein molecules are shown: Gly-17 and Asn-18 are from molecule 1; Asp- 142 and Ala-143 are from molecule 2; Glu-134 and Ser-135 are from a symmetry related copy of molecule 2. Carbon atoms are represented

to be hydrated. A consensus water-binding site, based on 16 examples observed in the L. casei and both E. coli molecules, lies about 2.8 A from the carbonyl oxygen, with a C-0-O(H20) angle of about 120" and a Ca-C-O-O(H20) dihedral angle of about 33". In effect the water molecule lies in a small pocket on the helix surface surrounded by the a and p carbons of the three helix residues n, n + 3, and n + 4, and is hydrogen bonded to the carbonyl oxygen of residue n. Several examples of water molecules that are hydrogen bonded in this manner to helix aB of the E. coli enzyme are depicted in Fig. 4.

Another interesting aspect of helix physiography shared by these two bacterial dihydrofolate reductases is the participa- tion of certain conserved amino acid side chains in helix termination. For example in both structures the hydrogen- bonding pattern of aB is terminated at Thr-34,lc (Thr-35,ec) with Oyl of the threonine donating a hydrogen bond to the carbonyl oxygen of Phe-30,lc (Phe-3l,ec), thereby replacing the threonine's backbone nitrogen atom. The backbone torsion angles of the residue preceding the threonine correspond to those of the a11 conformation (+ = -93", II/ = -18") associ- ated with helix termination by Nemethy et al. (1967), and the residue following the threonine has a nonhelical backbone geometry. Similarly, in both structures aC terminates immediately following Ser-48,lc (Ser-49,ec), of which the side chain Oy forms a hydrogen bond with the carbonyl oxygen from the preceding turn of the helix. Although our sample is admittedly very small, these examples suggest that the classification of serine and threonine as helix breakers (Levitt, 1978) is indeed related to side chain-to-backbone hydrogen bonding.

SECONDeRY STRUCTURE: HAIRPIN TURNS

Table I summarizes the location and geometry of the hairpin (reverse) turns in the L. casei and E. coli dihydrofolate reductase structures. For present purposes, we define a turn as consisting of any four residues n to n + 3 where the distance from Can to Cants is less than 7.0 A, subject to the restriction that both n and n + 3 are not part of the same a helix. Hairpin turns have been classified in several ways by other investiga- tors. A scheme suggested by Richardson (1981) recognizes types I, I', 11, 11', VIa, VIb, and IV from the set catalogued originally by Lewis et al. (1973). A turn belongs to one of the classes I, 1', 11, or 11' if the torsion angles (+,II/)n+l and are all within 50" of the ideal values derived by Venkatach- alam (1968). Turns incorporating a cis-proline residue a t PO-

by smaller open circles, oxygen atoms by larger open circles, and nitrogen atoms by blackened circles. Numbered circles represent f ied water molecules. Dashed lines indicate hydrogen bonds or metal-ligand interactions.

sition n + 2 are classified as type VIa or VIb according to the description given by Richardson (1981), whereas type IV is a miscellaneous category intended to include any turns not otherwise classified. Multiple turns (Isogai et al., 1980), i.e. those consisting of overlapping single turns, are grouped within Table I. The multiple turns beginning with residues 106,lc (103,ec), 132,lc (129,ec), and 148,lc are essentially equivalent to short 310 helices, each consisting of five residues.

In accord with our observation that the greatest structural differences between these two species of reductase lie in the loops connecting elements of a and ,B secondary structure, Table I shows that several turns present in the L. casei structure have different geometry in the E. coli molecule or are absent entirely. I t is interesting that the two extra turns in the L. casei structure, residues 51-54,lc and 65-68,1c, occur at points corresponding to single residue insertions in the L. casei sequence relative to that of the E. coli enzyme. The other two points of insertion and deletion, residues 91-92,lc and residue 148,lc, correspond to turns that exist in both structures but that differ markedly in backbone geometry.

Six of the nine insertions and deletions distinguishing the vertebrate reductases from the bacterial enzymes (Volz et al., 1982) coincide with reverse turns in the L. casei structure. These coincidences suggest to us that 1) formation of hairpin turns may provide a mechanism for accommodating single residue insertions and deletions in much the same manner as suggested by Richardson et al. (1978) with regard to ,8 bulges and 2) existing turns are points within the structure of a protein where further insertions can be tolerated.

CONFORMATIONAL FLEXIBILITY

Consistency of the observed variation in the isotropic thermal parameter, B, within the three refined crystallographically independent dihydrofolate reductase molecules suggests that, as expected, solvent-exposed loops have the greatest flexibility while the various elements of secondary structure are more rigid. In Fig. 5 we have plotted, versus residue number, the average of the individual atomic B factors for the backbone atoms of each residue in the L. casei structure and the two molecules in the E. coli structure, allowing equivalent residue numbers in the two species to coincide. Each of the three molecules shows essentially the same distribution of B values. Since the three molecules have markedly different environments within the two crystal structures, this similarity


FIG. 4. Water molecules hydrogen bonded to carbonyl oxygens of an a helix. A general pattern of helix hydration (described in the text) is exemplified by the binding of waters 609, 632, and 657 to helix aB of E. coli dihydrofolate reductase (molecule 2). Carbon atoms are represented by smaller open circles, oxygen atoms by larger open circles, and nitrogen atoms by blackened circles. Large numbered circles represent fixed solvent molecules. Hydrogen bonds between protein and solvent are indicated by dashed lines.

TABLE I Hairpin turns in bacterial dihvdrofolate reductases

L. cosei E. coli

Residues Sequence Type" H bondsh Residues Sequence TvDe H bonds

8-1 1 N-R-D-G I 08-Nll 9-12 A-V-D-R I1 15-18 K-D-G-H I' N15-018 16-19 M-E-N-A IV -

09-N12

015-N18 34-37 T-V-G-K I1 034-N37 35-38 T-L-D-K I1 51-54 K-R-P-L - 54-57 L-P-E-R I1 - 54-57 L-P-G-R I1 65-68 Q-E-D-Y I 70-73 A-Q-G-A I1 69-72 D-D-R-V I

IV 89-92 H-L-D-Q - 85-88 C-G-N-V IV 88-91 V-P-E-I IV

VIb -

Absent

Absent -

065-N68 070-N73 -

-

106-109 F-K-D-D I 0106-N109 103-106 F-L-P-K I 0103-N106 107-110 K-D-D-V I - 104-107 L-P-K-A I 0104-N107 132-135 N-W-D-D I 0132-N135 129-132 E-P-D-D I 0129-N132 133-136 W-D-D-F I 0133-N137 130-133 P-D-D-W I 146-149 D-T-N-P IV - 144-147 D-A-Q-N I N144-0147 148-151 N-P-A-L I 0148-NE1 149-152 P-A-L-T I -

-

-

The definition of what constitutes a turn and the scheme for classifying the turns as to type are given in the text. Turns that share one or

'We list only those hydrogen bonds where both donor and acceptor are main chain atoms from the four residues within the turn. A dashed more residues are grouped together.

line indicates that there are no hydrogen bonds of this kind.

must reflect a property of the enzyme structure itself. Artym- iuk et al. (1979) have drawn the same conclusion based on a similar comparison of the thermal parameter distribution in human and egg white lysozyme, for which 60% of the amino acid residues are identical. Our comparison between the E. coli and I,. casei dihydrofolate reductases extends their conclusion to a pair of protein structures with significantly less sequence homology (27% identity).

A major problem confronting detailed interpretation of thermal parameters in terms of molecular motion is the ina- bility of the crystallographic experiment to distinguish true thermal motion from static disorder within the crystal. Indeed, five residues in the E. coli structure (Ser-64,ecl, Ser-l5O,ecl,

His-45,ecl, Ser-64,ec2, and Asp-l22,ec2) that had high side chain B values in early stages of the refinement were later recognized to be disordered between two different side chain conformations. In all five cases both conformations are stabi- lized by hydrogen bonding, and both correspond to one or another of the preferred conformations for that type of side chain obtained from analysis of numerous other protein structures (Janin et al., 1978). It is noteworthy that three of the five are serines and that a similar situation was found in the refined structure of actinidin (Baker, 1980). Serine is probably a candidate for conformational disorder in all protein structures because of its ability to serve either as a hydrogen bond donor or as an acceptor and because of its low barrier to


molecule 1

5 0 -

10 -

I 20 40 60 80 100 120 140

Residue number

70 E.ca/i O H F R L

I I I 20 40 60 eo 100 120 140

Resldue number

70 L case, DHFR L

2 1 50 I t I I

I 20 40 60 80 100 120 140 160

Resldue number

FIG. 5. Isotropic thermal parameters (B) of backbone atoms in dihydrofolate reductase (DHFR). For each residue the individual temperature factors of N, Ca, C, and 0 have been averaged. Structurally equivalent residues are aligned vertically in the three panels. Gaps in the plot correspond to insertions/deletions.

rotation about the Ca-Cp bond (Janin et al., 1978).

ADDITIONAL COMMENTS CONCERNING BOUND SOLVENT

The asymmetric unit of the L. casei dihydrofolate reductase crystal structure contains altogether approximately 1200 water molecules. Of these a total of 264 have been located in fixed positions and their coordinates, occupancies, and temperature factors refined. The corresponding numbers for the E. coli dihydrofolate reductase crystal structure are about 800 water molecules per asymmetric unit, of which 428 are located and refined. Additionally, two chloride ions and a calcium ion are included in the list of bound solvent molecules in the E. coli structure.

In both species the overall picture of the fixed solvent structure corresponds to a monomolecular hydration shell bound to the exposed surface of the protein molecule, with about 60 to 70% of the water oxygens hydrogen bonded to at least one protein nitrogen or oxygen atom. Those remaining fixed water molecules that are not hydrogen bonded directly to protein atoms instead link up with other water molecules to form short bridges, 3 to 5 molecules long, from one hydrogen-bonding protein group to another. Some bridging water molecules are within 3.5 A of a nonpolar protein group, while others are further from the protein surface, almost always when they occupy large crevices on the surface or gaps between different protein molecules. Every fixed water molecule is hydrogen bonded to something, and the entire structure of the hydration shell is organized around protein polar groups.

There are no clathrate-like water cages surrounding nonpolar groups.

To what extent are these fixed waters an invariant conserved feature of dihydrofolate reductase architecture? To begin with, we compare the two molecules in the crystallographic asymmetric unit of the E. coli enzyme structure which are, by definition, unrelated by symmetry and thus see different environments. In this case, we observe that only 50% of the fixed waters that are hydrogen bonded directly to each molecule (and 60% of the waters hydrogen bonded to the main chain) occupy the same sites in both. Waters were considered to occupy equivalent sites if they were less than 1 A apart when the two solvated molecules of the asymmetric unit were superimposed and were also hydrogen bonded to corresponding protein atoms. (Although this criterion is fairly rigorous, the percentages would not increase appreciably if the distance requirement were relaxed to about 1.5 A.) Thus it is clear that the bound water structure depends on details of the enzyme molecule’s surroundings and is influenced by intermolecular contacts in the crystal. A similar observation has been made regarding the bound water in triclinic as compared with te- tragonal hen egg white lysozyme (Moult et al., 1976).

Focusing next on a comparison between the bound waters of L. casei and E. coli dihydrofolate reductases, the waters that might be similarly bound in both structures may for convenience be divided into two groups. Fixed water molecules in the fiist group are simultaneously hydrogen bonded to the enzyme and to the methotrexate inhibitor or the NADPH cofactor. Two protein-bound waters can be identified that are also hydrogen bonded to the methotrexate and identically situated in both species of enzyme, as will be seen later when we describe the binding of the inhibitor. Furthermore, as discussed in the following paper, seven more water molecules mediate the binding of NADPH in the L. casei ternary complex; for these seven waters no direct comparison with waters bound to the E. coli binary complex is possible.

The second comparison group of potentially conserved bound waters might be characterized as purely structural, that is, water molecules that function to tie together elements of protein structure. Out of a total of 178 water molecules bound directly to the L. casei enzyme, 65 are simultaneously hydrogen bonded to two or more protein groups and are thus candidates for this role. However, only three of these waters were found that have at least two protein ligands in common in both E. coli molecules and also in the L. casei molecule. One of them, Wat5-255,lc (Wat-530,ecl; Wat-608,ec2), stabi- lizes the local conformation at the COOH-terminal end of (YB by linking the carbonyl oxygens of residues 32 and 35,lc (33 and 36,ec). A second, Wat-263,lc (Wat-422,ecl; Wat-635,ec2) bridges a gap at the NH2-terminal edge of the p sheet by hydrogen bonding to both the peptide nitrogen of Asn-59,lc (Asn-59,ec) and the carbonyl oxygen of Gly-72,lc (Arg-71,ec) at the point where the loops leading into PC and @D diverge. Neither of these conserved bound water molecules is internal to the protein structure, and in fact there are no strictly internal water molecules in either bacterial dihydrofolate reductase, if we eliminate from consideration a few water molecules that are trapped by cofactor or inhibitor binding (three in the L. casei s€ructure and one in the E. coli structure).

The third water molecule in this group, Wat-201,lc (Wat- 405,ecl; Wat639,ec2) also participates in methotrexate binding (see below) and is the single “trapped” water common to both bacterial reductase structures. In both structures, this water molecule hydrogen bonds to the conserved residue Thr-116,lc (Thr-113,ec) and the 2-amino group of methotrexate and is ’ The abbreviation used is: Wat, water.


within hydrogen-bonding distance of three other potential ligands, at least two of which are protein atoms. We suspect that if Wat-201,lc has any role in protein folding, it is subor- dinate to the role this water plays in pteridine binding.

THE METHOTREXATE-BINDING SITE

From our previous unrefined crystallographic results on dihydrofolate reductase we already know that the conformation of the bound methotrexate molecule and its interactions with the enzyme are very similar in the E. coli binary and L. casei ternary complex (Matthews et al., 1977, 1978). As an expected correlation, amino acid sequence comparisons among the known dihydrofolate reductases (Volz et al., 1982) show that most of the residues involved in methotrexate binding are highly conserved. This general picture has not changed with crystallographic refinement, but several interesting details have emerged and a number of significant revisions have resulted, notably with respect to the role of fixed water molecules in the binding of methotrexate. In the interest of making this section easier to follow it should be mentioned here, in anticipation of a more detailed discussion, that we believe the pteridine ring of a bound substrate will be turned upside down from the way it appears in the bound methotrexate, but that the surrounding hydrogen-bonding groups, both protein and fixed solvent, will retain the same geometry in the enzyme-substrate complex.

Pteridine- binding Site (Specific Hydrogen Bonding)-Fig. 6 shows the 2,4-diaminopteridine ring of methotrexate and its immediate surroundings in the L. casei enzyme. Unless otherwise noted, all features described in this section are identical in both bacterial enzyme structures. A variety of evidence, spectroscopic (reviewed by Gready, 1980; see also Cocco et al., 1981a, 1981b; and Ozaki et al., 1981), calorimetric (Subraman- ian and Kaufman, 1978), and theoretical (Perault and Pull- man, 1961), strongly suggests that N1 is protonated when methotrexate is bound, and in the following discussion we assume this to be the case.

In our original interpretation (Matthews et al., 1978) we had erroneously positioned thy pteridine ring slightly to the left, relative to its location in Fig. 6, so that Asp-26,lc appeared to interact with three groups, the 2-amino group, N1, and N8. As a result of refinement we now see that the interaction is simpler, with OS1 hydrogen bonded to the 2-amino group and 062 hydrogen bonded to N1, and with N8 too far from either

aspartate oxygen to form a hydrogen bond. Since N1 is protonated and presumably Asp-26,lc is anionic, this interaction must also be ionic in nature. Although it is conserved in the bacterial sequences, the buried negatively charged residue Asp-26,lc (Asp-27,ec) is replaced by Glu-30 in the vertebrate sequences; however, Volz et al. (1982) have shown that Glu- 30 in the chicken liver enzyme makes an analogous interaction with a bound inhibitor heterocycle.

As a result of this slight misplacement of the pteridine ring our earlier model was constructed with a direct hydrogen bond between the 2-amino group and Thr-116,lc (Thr-l13,ec), whereas in the refined structures we now see that a fixed water molecule, Wat-201,lc (Wat-405,ecl; Wat-639,ec2), lies between the 2-amino group and the Thr-116,lc side chain. Since this threonine is strictly conserved in all dihydrofolate reductase sequences, we suspect that the bridging water molecule is an also invariant feature of enzyme structure. Note that 061 of Asp-26,lc is also hydrogen bonded to Thr-llG,lc, so that the triad Asp-26,lc, Thr-l16,lc, and Wat-201,lc forms a tightly constrained polar pocket complementary in geometry and charge distribution to the three protons on N1 and the 2- amino group. We will also assume that similar complementar- ity is a feature of the enzyme-substrate complex when we describe a hypothetical model for this complex in the next section.

In our original unrefined models the 4-amino group of methotrexate donated only a single hydrogen bond, namely to the carbonyl oxygen of Leu-4,lc. Crystallographic refinement, however, has revised the backbone geometry of residues 97-100,lc (it now includes a cis peptide bond between Gly-98 and Gly-99, as mentioned above) with the result that a second hydrogen bond is now evident, from the 4-amino group to the carbonyl oxygen of Ala-97,lc. Moreover no other potential hydrogen bond donor, protein or solvent, is present near the 4-amino group. Thus the binding site occupied by the 4-amino group of the methotrexate inhibitor is apparently designed to accommodate some hydrogen bond-donating atom of the substrate. We will suggest below that this atom could be N8.

In contrast to the pyrimidine portion of the methotrexate pteridine ring, its pyrazine portion is not directly hydrogen bonded to the enzyme a t all (see Fig. 6). Instead, N5 makes no hydrogen bonds of any kind, while N8 is weakly hydrogen bonded only to a structurally conserved fixed water molecule, Wat-253,lc (Wat-403,ecl; Wat-603,ec2), which lies at a dis-

FIG. 6. The pteridine-binding site of L. casei dihydrofolate reductase. Methotrexate is indicated by solid

portion of the NADPH molecule by bonds, protein by open bonds, and a

striped bonds. Carbon atoms are represented by smaller open circles, oxygen atoms by larger open circles, and nitrogen atoms by blackened circles. Large numbered circles represent fixed solvent molecules. Hydrogen bonds, indicated by dashed lines, are listed in Table 111 and described in the text.


tance of 3.3 A from N8, directly in line with its lone pair orbital. In turn Wat-253,lc hydrogen bonds to OS2 of Asp- 26,lc, to another conserved fixed water Wat-217,lc (Wat- 412,ecl; Wat-604,ec2), and to Ne1 of Trp-21,lc (Trp-22,ec). Finally, Wat-217 is hydrogen bonded to the backbone amide nitrogen of Leu-23,lc (Leu-24,ec). Both the tryptophan and leucine residues are strictly conserved, although it is not yet clear why, since neither appears to interact strongly with either inhibitor or cofactor. The only difference in this region of the structure between the E. coli and L. casei enzymes is that the hydrogen bond between Wat-253,lc and Ne1 of Trp- 21,lc is not a direct one in the E. coli enzyme but instead is mediated by yet another fixed water (Wat-567,ecl; Wat- 702,ec2), connecting the first fixed water with the tryptophan residue. Evidently this variation reflects a slight difference in backbone conformation between the two bacterial enzyme structures.

Pteridine- binding Site (Nonpolar Interactions)-Nonpolar interactions between the pteridine ring and the enzyme remain essentially the same as reported by Matthews et al. (1978) prior to refinement, with the side chains of residues 4, 6, 27,30, and 97 (in the L. casei numbering system) providing most of the van der Waals contacts. Among these residues only Ala-6,lc and Phe-30,lc are strictly conserved, although all of them are hydrophobic in all the known sequences. The backbone chain of Trp-5,lc and Ala-6,lc also remains in contact with the pteridine ring, but contrary to our prerefinement suggestion, the 5-6,lc peptide bond is not parallel to the pteridine ring and does not make a 7-n interaction with it.

In the L. casei ternary complex additional nonplar contacts with the pteridine ring are made by the side chain of Leu-19,lc and by the nicotinamide ring, which lie against the face of the pteridine ring adjoining the nicotinamide-binding site, isolat- ing it from the solvent. In contrast, the residue corresponding to Leu-19,lc in the E. coli binary complex, namely Met-20,ec, is folded away from the inhibitor, leaving this face of the pteridine ring exposed. The otherwise unoccupied nicotinamide-binding site is filed instead with fived water molecules linked to the protein and to one another by a network of hydrogen bonds. However, none of these water molecules forms a specific hydrogen bond with any part of the bound methotrexate molecule.

These additional contacts between the methotrexate and

the nicotinamide in the ternary complex, together with the accompanying desolvation of the exposed face of the pteridine ring on binding of the nicotinamide, may explain the enhanced binding of methotrexate to the enzyme:NADPH binary complex (the holoenzyme) relative to the apoenzyme (Poe et al., 1974; Williams et al., 1979).

Binding of the p-Aminobenzoyl Group-The p-aminobenzoyl moiety of methotrexate occupies a hydrophobic pocket formed by the helix uB on one side and by the loop (50-56,lc) connecting uC to PC on the other. In particular, the side chains of Leu-27,lc and Phe-30,lc (Leu-28,ec and Phe-31,ec) extending from uB interact with the inner face of the p- aminobenzoyl group. This phenylalanine, but not the leucine, is strictly conserved in all known sequences. On the opposite side of the pocket the interactions, which have had to be somewhat revised as a result of refinement, differ appreciably between the two bacterial enzyme structures. The interactions between methotrexate's p-aminobenzoyl group and the L. casei enzyme are shown in Fig. 7.

In the case of the E. coli binary complex, the side chains of Ile-50,ec, Leu-54,ec, and Ile-94,ec make van der Waals contact with the outer face of the p-aminobenzoyl group, as in our original prerefinement model. Of these three residues only the leucine is conserved in all dihydrofolate reductase sequences. A new feature, brought out by refinement, is a set of hydrogen bonds involving the carbonyl oxygen of the benzoyl moiety. The side chain nitrogen atom Nq2 of Arg-52,ec and a fixed water molecule, Wat-566,ecl or Wat-685,ec2, are directly coordinated to the latter, while simultaneously, the water molecule is hydrogen bonded to the carbonyl oxygen of Ile-50,ec and to NE of Arg-52,ec.

In the case of the L. casei ternary complex, refinement has resulted in significant revision of the geometry of residues 49- 54,lc (including introductior! of a cis peptide bond a t Arg-52- Pro-53,lc as mentioned earlier) and consequent revision of the interaction between this chain segment and the p-aminobenzoyl group. The side chain of Phe-49,lc (corresponds to Ile- 50,ec) is in van der Waals contact, but its benzene ring is now seen to be inclined at an angle of 45" to the p-aminobenzoyl ring, eliminating the possibility of the ring-stacking interaction we had earlier suggested. Other hydrophobic interactions include those with the side chains of Pro-50,lc and Leu-54,lc.

Additionally, the immediate environment of the benzoyl

FIG. 7. The binding of methotrexate to L casei dihydrofolate sented by smaller open circles, oxygen atoms by larger open circles, reductase. Interactions between the enzyme and the p-aminoben- and nitrogen atoms by blackened circles. Large numbered circles zoyl-L-glutamate portion of the inhibitor are emphasized. Methotrex- represent fixed solvent molecules. Hydrogen bonds, indicated by ate is indicated by solid bonds, protein by open bonds, and a portion dashed lines, are listed in Table 111 and described in the text. of the NADPH molecule by striped bonds. Carbon atoms are repre-


group's carbonyl oxygen is quite different in the L. casei complex. Instead of hydrogen bonding with an arginine side chain, its nearest neighbors are two fixed water molecules, Wat-322,lc and Wat-606,lc, which are apparently otherwise uninvolved with the protein structure. The side chain of Arg- 52,lc, meanwhile, is pointed in the opposite direction and interacts with the backbone carbonyl oxygen of Phe-49,lc.

Binding of the Glutamyl Carboxylates-In both refined dihydrofolate reductase structures binding of the glutamate portion of methotrexate (see Fig. 7) is dominated by a pair of hydrogen bonds between its a-carboxylate and the guanidinium group of Arg-57,lc (Arg-57,ec), a strictly conserved residue. Although this ion pair lies near the molecular surface, it is sequestered from the solvent in a narrow pocket formed by aliphatic carbons of nearly side chains, and we can identify several hydrogen bonds that appear to control the conformation of Arg-57,lc, forcing the guanidinium group into this hydrophobic environment. Specifically, the guanidinium group is rigidly oriented by hydrogen bonds to O y l of Thr- 34,lc, a conserved residue, and to the carbonyl oxygen of Pro- 55,lc. Three additional hydrogen bonds nearby also restrict local flexibility, namely those between the side chain amide of Asn-59,lc (conserved in six of seven sequences) and the backbone of residues 54 and 57,lc. Significantly, Arg-57,ec main- tains the same conformation in the refined enzyme-trimethoprim complex6 as in the E . coli enzyme-methotrexate complex despite the absence in trimethoprim of any substituent analogous to methotrexate's glutamyl a-carboxylate group.

In contrast to the binding of the glutamyl a-carboxylate, binding of the y-carboxylate appears to be weak and nonspecific, and it differs between the L. casei and the E. coli structures. In the L. casei complex, His-28,lc hydrogen bonds to one of the y-carboxylate oxygens, but the thermal parameters of the glutamate side chain are large and vary widely (B = 30 to 55 A2), indicating disorder. The y-carboxylate in the E . coli complex, on the other hand, interacts only with several water molecules that are hydrogen bonded to Lys-32,ec and to the inhibitor's a-carboxylate and benzoylcarbonyl oxygen. In correction of our pre-refinement model, it should be mentioned here that the refined E. coli structure shows that the y-carboxylate is bound in the same manner in both independent molecules and also shows that this inhibitor group does not hydrogen bond directly to Lys-32,ec or any other protein residue. Probably this same nonspecific binding also occurs in the association between bacterial dihydrofolate reductases and folyl-polyglutamate substrates, since, a t pH 7 the disso- ciation constants of these species are not significantly different from that of the monoglutamate form (Blakley et al., 1982).

In Tables I1 and I11 we have summarized the hydrogen bonding interactions and the nonpolar interactions between methotrexate and both species of reductase.

A MODEL FOR SUBSTRATE BINDING

In our earlier report on the structure of L. casei dihydrofolate reductase (Matthews et al., 1978) we noted that, although methotrexate binds with its pteridine ring's C7-re face turned toward the cofactor nicotinamide moiety, the binding site appears capable of accommodating a pteridine ring with either face adjacent to the nicotinamide while keeping C6 of the pteridine near C4 of the nicotinamide. Pursuing this idea, we proposed that a large body of evidence (reviewed by Gready, 1980) suggesting that methotrexate binds somewhat differently from folate and dihydrofolate might be explained if substrate does in fact bind with its pteridine ring rotated by 180" about the C6"C9 bond. We further noted that our model predicts an absolute configuration of R at C6 for the product

" J. T. Bolin and C. Beddell, personal communication.

tetrahydrofolate if substrate dihydrofolate is bound like methotrexate, but S if its pteridine ring is turned over. Subse- quently Fontecilla-Camps et al. (1979) showed by x-ray diffraction analysis that 5,lO-methenyltetrahydrofolate derived from the biologically active diastereoisomer of folinic acid indeed does have the absolute configuration S a t C6. Soon after, Charlton et al. (1979) established the absolute stereochemistry of folic acid reduction by dihydrofolate reductase, showing that reduction at either C6 or C7 involves formal hydride transfer of the 4-pro-R (A side) hydrogen of NADPH to the same (C7-si) face of folic acid. Thus, to summarize, both folic acid and dihydrofolate must bind to the enzyme in some conformation that presents the opposite face of the pteridine ring to the nicotinamide, relative to the orientation of the pteridine ring of bound methotrexate.

In an attempt to define the principal interactions between dihydrofolate reductase and the pteridine ring of a substrate, we have modeled the fit of the 4-keto tautomer of 7,8-dihydrofolate to the methotrexate-binding site of both refined enzyme structures. The 4-keto tautomer is known to be the stable form of dihydrofolate, both in solution (Pfleiderer et al., 1960; Brown and Jacobsen, 1961) and when bound to the enzyme (Hood and Roberts, 1978). In this modeling experiment we were guided by the following simplifying assump- tions: 1) the conformation and solvation of the protein in the enzyme:substrate complex are the same as in the enzyme:methotrexate complex; 2) the binding of the p-aminobenzoyl-L-glutamate portion of the substrate is identical with that of the inhibitor; and 3) the substrate has C7-si face of its pteridine ring turned toward the nicotinamide-binding site and will maximize its hydrogen bonding to the enzyme. In other words we simply replaced the inhibitor pteridine ring with a 2-amino-4-oxo dihydropteridine and turned it over by rotating about only the C6-C9, C9-Nl0, and NlO"C4' bonds. We find that a very reasonable model can be built in this way, with C6 about 3.5 A from the nicotinamide C4.

Fig. 8a shows the resulting hypothetical model for binding of the substrate pteridine ring. It may be compared with Fig. 86, which shows the bound pteridine ring of methotrexate. The model predicts that, as in the methotrexate complex, the 2-amino group of the substrate will form two hydrogen bonds, one with 081 of Asp-26,lc and the second with Wat-2Ol,lc, but that rotation of the pteridine ring will position N3, instead of N1, within hydrogen-bonding distance of 062. Moreover, as a result of this rotation the position of the 4-oxo substituent in the hypothetical enzyme:substrate complex corresponds to that of N8 in the methotrexate complex, permitting formation of a hydrogen bond between the 4-oxo group and Wat-253,lc. Simultaneously N8 of the substrate takes the place of the 4- amino group on methotrexate so that the hydrogen atom on N8 can hydrogen bond either to the carbonyl oxygen of Leu- 4,lc, or to that of Ala-97,lc. I t should be noted that this model predicts the net loss of one protein-ligand hydrogen bond in the dihydrofolate complex, relative to the binding of methotrexate in its complex, as a result of replacing the inhibitor's 4-amino group with N8 of the substrate. Although Fig. 8a depicts a hydrogen bonding arrangement in which Asp-26,lc is negatively charged, we emphasize that Asp-26,lc may in fact be protonated. An additional proton could be accommodated by rearranging the hydrogen bonds involving either Wat- 253,lc or Thr-116,lc and Wat-201,lc. In both possible protonation states Asp-26,lc can make to two hydrogen bonds with N3 and the 2-amino group of the substrate and a third hydrogen bond with Wat-253,lc.

Our substrate-binding model differs significantly from the model proposed by Hitchings and Roth (1980). In the process of rotating the pteridine ring from the methotrexate orienta-


N10-methyl

p-Aminobenzamide

TABLE I1 Hydrophobic and van der Waals interactions between methotrexate and dihvdrofolate reductase

Component of methotrexate Contact in L. casei DHFR" Contact in E. coli DHFR Sequence conservationh

6-Me-Pteridine Leu-4 side chain Ile-5 side chain Trp-5 main chain Ala-6 main chain Ala-6 N Ca Cp Ala-7 N Ca Cp Conserved Leu-19 side chain var. bound waters ex. E. coli (Met) Leu-27 side chain Leu-28 side chain Phe-30 side chain Phe-31 side chain Conserved Ala-97 Ca Cp Ile-94 Ca Cp Nicotinamide ring var. bound waters Leu-19 side chain var. bound waters ex. E. coli (Met) Ser-48 side chain Ser-49 side chain Wat-439 var. bound waters Leu-27 side chain Leu-28 side chain Phe-30 side chain Phe-31 side chain Conserved Phe-49 side chain Ile-50 side chain Pro-50 side chain Leu-54 side chain Leu-54 side chain Conserved

L-Glutamate Leu-27 side chain Leu-28 side chain Phe-30 side chain Phe-31 side chain Conserved Arg-31 Cp Cy C6 Lys-32 cp c y cs Leu-54 side chain Leu-54 side chain Conserved

ex. S. faecium (Ala)

" The abbreviations used are: DHFR, dihydrofolate reductase; var., various; ex., except. ' Only those residues that are identically conserved in all known genomic sequences (according to the alignment of Volz et al., 1982) are

listed as "conserved." For residues that are identically conserved in all but one species, the exception is listed.

TABLE I11 Hydrogen- bonding interactions between methotrexate and dihydrofolate reductase

Component of methotrexate Contact in L. casei DHFR" Contact in E. coli DHFR Description

Pteridine, N1 and 2-amino Asp-26 carboxylate Asp-27 carboxylate Two H Bonds; ionic; always Asp

2-Amino group Wat-201 Wat-405; (Wat-639) Water also bound by conserved

N8 Wat-253 Wat-403; (Wat-603) (long con- Water also bound by Asp-26,lc

4-Amino group Leu-4 and Ala-97 carbonyl Ile-5 and Ile-94 carbonyl groups

Benzoylcarbonyl Arg-52 guanidinium, Wat-566; Water also bound by Arg-52,ec

L-Glutamate, a-carboxylate Arg-57 guanidinium y-Carboxylate His-28 imidazole

or Glu

Thr 116,lc

tact) and conserved Trp-21,lc

groups

(Wat-685) Arg-57 guanidinium Various waters

and carbonyl 50,ec Conserved Arg

DHFR, dihydrofolate reductase.

a b FIG. 8. Schematic representation of hydrogen bonding between dihydrofolate reductase and the pteridine portions of a, 7.8-dihydrofolate

A L A 97 (hypothetical) and b, methotrexate. "R" represents p-aminobenzoyl-L-gluta-

to the sequence of the L. casei enzyme. The interactions with methotrexate (6) are observed in high resolution crystal-

dihydrofolate reductases. Interactions L E U ' with dihydrofolate ( a ) are predicted by

model-building experiments described in the text. The unlabeled carbonyl oxygen

enzyme-substrate complex might be

253 ..___... y 0 =( mate. Residues are numbered according

0 ______._ H-y '',~oq lographic studies of E. coli and L casei !

0 """" H"

! n li H y , ; ____--. H - / - H _ _ _ _ _ _ 0 yi ______. H - b - H - _ _ _ _ _ 0 ==( hydrogen bonded to N8 in the proposed

201 20 1

THR 116 L E U I14 T H R 1 I6 L E U I 1 4 Leu-4, Ala-97, or both.

tion to that of the substrate we exchange the positions of N1 and N3, whereas Hitchings and Roth appear to exchange N1 and C4. As a result, in the latter model the 2-amino group is displaced from its position adjacent to Asp-26,lc, and the side chain of this residue lies near N5 and the 4-oxo group of the substrate instead of forming hydrogen bonds with N3 and the 2-amino group. The same is true of the model described by Williams and Morrison (1981), which is evidently indistin- guishable from that of Hitchings and Roth. Although the Hitchings-Roth model would be appealing in that it does

suggest how protonation at N5 could be promoted, it should be emphasized that we cannot construct such a model without generating several hopelessly short nonbonded contacts between the substrate and the enzyme. In fact, we have never, in our model-building experiments, been able to place N5 near Asp-26,lc without requiring an extensive and unpredictable change in protein conformation or without violating our as- sumption that the substrate's p-aminobenzoyl-L-glutamate group is bound in the same manner as that of methotrexate.

The model we have proposed here qualitatively predicts


the relative magnitudes of the binding constants for methotrexate, dihydrofolate, and folate. Based on fluorescence- quenching data, Dann et al. (1976) determined the association constant for 7,8-dihydrofolate with the L. casei enzyme to be 2 X lo6 M", while Hood and Roberts (1978) and Cocco et al. (1981b) have estimated the association constant between the L. casei enzyme and the N1-protonated form of methotrexate to be approximately 2 X 10". According to the above model, this factor of lo7 difference is due to 1) an additional pteridine- to-protein hydrogen bond involving the 4-amino group in the methotrexate complex and 2) favorable charge-charge interaction between Asp-26,lc and the N1-protonated pteridine of methotrexate.

Our model also predicts that if folate and 7,8-dihydrofolate bind with the same geometry, the former compound will have a smaller association constant than the latter because of the absence of a hydrogen bond between N8 and the carbonyl oxygen of Leu-4,lc or Ala-97,lc in the folate complex. This prediction is in general agreement with the relative magnitudes of observed binding constants for folate and dihydrofolate in binary complexes with both the L. casei (Dann et al., 1976) and E. coli enzymes (Greenfield et al., 1972; Cayley et al., 1981), though in the latter species the difference was found to be less than a factor of 10.

Implications of the substrate-binding model depicted in Fig. 8a for the catalytic mechanism will be discussed in the following paper.

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