Atomic Structures of Human Dihydrofolate ReductaseComplexed with NADPH and Two LipophilicAntifolates at 1.09 A and 1.05 A Resolution

Anthony E. Klon1, Annie Heroux2, Larry J. Ross2, Vibha Pathak2,Cheryl A. Johnson2, James R. Piper2 and David W. Borhani2*

1Department of Biochemistryand Molecular GeneticsUniversity of Alabama atBirmingham, Birmingham, AL35294, USA

2Drug Discovery DivisionSouthern Research InstituteBirmingham, AL 35205, USA

The crystal structures of two human dihydrofolate reductase (hDHFR)ternary complexes, each with bound NADPH cofactor and a lipophilicantifolate inhibitor, have been determined at atomic resolution. The potentinhibitors 6-([5-quinolylamino]methyl)-2,4-diamino-5-methylpyrido[2,3-d ]pyrimidine (SRI-9439) and (Z )-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d ]pyrimidine (SRI-9662) were developedat Southern Research Institute against Toxoplasma gondii DHFR-thymidyl-ate synthase. The 5-deazapteridine ring of each inhibitor adopts anunusual puckered conformation that enables the formation of identicalcontacts in the active site. Conversely, the quinoline and dimethoxy-benzene moieties exhibit distinct binding characteristics that account forthe differences in inhibitory activity. In both structures, a salt-bridge isformed between Arg70 in the active site and Glu44 from a symmetry-related molecule in the crystal lattice that mimics the binding ofmethotrexate to DHFR.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: lipophilic antifolates; distorted geometry; inhibitor selectivity*Corresponding author

Introduction

Dihydrofolate reductase (DHFR; EC 1.5.1.3)catalyzes the reduction of 7,8-dihydrofolate orfolate to 5,6,7,8-tetrahydrofolate in an NADPH-dependent manner. Tetrahydrofolate is an essentialcofactor for a number of enzymes catalyzing one-carbon-transfer reactions necessary for the bio-synthesis of numerous amino acids and purines.Tetrahydrofolate acts also as a cofactor forthymidylate synthase, which catalyzes the methyl-ation of deoxyuridylate to thymidylate, a key com-ponent of DNA. DHFR is found in cells of all livingorganisms, where it maintains the intracellularlevel of reduced folates. Therefore, the inhibitionof DHFR activity reduces the intracellular pool of

tetrahydrofolate, resulting in inhibition of DNAsynthesis, eventually leading to cell death. As aresult, human DHFR (hDHFR) has become amajor drug target in anticancer therapy, as havethe bacterial, fungal, and protozoal DHFRs of anumber of microorganisms implicated in humaninfectious diseases.1,2

Understanding the structural basis of inhibitorbinding to hDHFR is a critical step in the designof inhibitors selective for microbial versus hDHFR.Towards this end, atomic resolution crystal struc-tures can reveal the subtle differences betweenmicrobial DHFRs and hDHFR, facilitating thedesign of inhibitors with increased efficacy andselectivity.

We present here the atomic-resolution crystalstructure of two ternary complexes of hDHFRwith its cofactor NADPH and two different inhibi-tors bound in the active site. To our knowledge,this is the first example of a DHFR crystal structureat atomic resolution. The electron density mapsallowed unambiguous placement of the NADPHcofactor as well as the inhibitors in each structure.In addition, the high resolution of the data allowsus to discuss the detailed geometry of each of theinhibitors and the NADPH cofactor in their bind-ing to the active site.

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

Present address: D. W. Borhani, Abbott BioresearchCenter, 100 Research Drive, Worcester, MA 01605, USA;A. Heroux, Brookhaven National Laboratory, Upton, NY11973, USA.

E-mail address of the corresponding author:david.borhani@abbott.com

Abbreviations used: DHFR, dihydrofolate reductase;hDHFR, human DHFR; tgDHFR, T. gondii DHFR;lmDHFR, Leishmania major DHFR; TS, thymidylatesynthase; DMSO, dimethylsulfoxide; PTX, piritrexim;TMQ, trimetrexate.

doi:10.1016/S0022-2836(02)00469-2 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 320, 677–693

Results and Discussion

SRI-9439 and SRI-9662 are potentDHFR inhibitors

SRI-9439 and SRI-9662 were designed atSouthern Research Institute as potent (DHFR)inhibitors of Toxoplasma gondii DHFR-thymidylatesynthase (DHFR-TS).3 These lipophilic antifolates,analogues of the DHFR substrate folic acid, are 6-([5-quinolylamino]methyl)-2,4-diamino-5-methyl-pyrido[2,3-d ]pyrimidine (SRI-9439), and (Z )-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d ]pyrimidine (SRI-9662) (Figure1). The IC50 values of SRI-9439 against T. gondiiDHFR-TS and rat liver DHFR were determined tobe 8 nM and 7 nM, respectively (selectivity ratio of0.88; [DHF] ¼ 92 mM, [NADPH] ¼ 120 mM). TheIC50 values of SRI-9662 against T. gondii DHFR-TSand rat liver DHFR were 12.3 nM and 112 nM(selectivity ratio of 9.1). SRI-9662 is the immediateprecursor to another potent inhibitor, SRI-7894.SRI-7894 is structurally identical with SRI-9662,with the exception that the cis double bond in thelinker region has been reduced to a single bond.SRI-7894 exhibits an IC50 of 7.9 nM against T. gondiiDHFR-TS and 770 nM against rat liver DHFR(selectivity ratio of 97.5).3

Structure determination

Both the SRI-9439 and SRI-9662/NADPH/hDHFR complexes crystallized in the ortho-rhombic space group P212121 with one complexper asymmetric unit. X-ray diffraction data foreach hDHFR ternary complex were collected froma single crystal. The SRI-9439 ternary complexcrystal diffracted X-rays to 1.09 A resolution, anddata from the SRI-9662 ternary complex extendedto 1.05 A. Both structures were solved usingmolecular replacement, and the NADPH andinhibitors in both structures were built intounambiguous electron density. Individual aniso-tropic temperature factors were refined and ridinghydrogen atoms were placed at their calculatedpositions. Restraints were removed from mostinhibitor atoms and some NADPH atoms in theSRI-9439 complex (from the pyrophosphatethrough the nicotinamide ring) in the final cyclesof refinement, resulting in final Rfree factors of17.3% for the SRI-9439 complex and 18.1% for theSRI-9662 complex.

The model for the hDHFR/NADPH/SRI-9439ternary complex includes 184 amino acid residues,one molecule each of NADPH and SRI-9439, twosulfate anions, and 436 water molecules. TheN-terminal valine and glycine residues had poor

Figure 1. Structures of lipophilic antifolates and folic acid. The atoms in the chemical structures of the antifolatesused in this study, SRI-9439 and SRI-9662, are numbered. The structures of SRI-7894 (the reduction product of SRI-9662), piritrexim (PTX), trimetrexate (TMQ), and folic acid are shown for reference.

678 Atomic Resolution Human DHFR Structures

electron density, and so were not modeled. Themodel for the hDHFR/NADPH/SRI-9662 ternarycomplex includes 185 amino acid residues, onemolecule each of NADPH and SRI-9662, onesulfate anion, one molecule of dimethylsulfoxide(DMSO), and 338 water molecules. The NADPHcofactor is modeled in the active site at 70%occupancy, and the DMSO molecule is bound atthe nicotinamide site at 30% occupancy. TheC-terminal aspartate residue had poor electrondensity and was not modeled. The data collectionand refinement are summarized in Table 1. Thetwo models are very similar, with an rms deviationof 0.20 A for 179 Ca atoms (out of 182 aligned Ca

atoms total, 1 A cutoff).A number of side-chain atoms in both models

were set to zero occupancy because either there

was no visible density or the electron density mapwas too ambiguous to allow their placement.Furthermore, residues Arg77 and Glu78 were sub-stantially disordered. There was only weak densityfor several of the side-chain atoms, and the densityfor the backbone in this region, although present,did not allow for unambiguous placement.Although refinement of the main-chain atoms athalf occupancy resulted in a lower Rfree factor thanrefinement with the backbone atoms set to eitherfull occupancy or no occupancy, coordinates forthese residues should be treated with caution.

Overall structure

The overall structure of the ternary complexmostly agrees with previously published models

Table 1. Data collection and refinement statistics

Data collection SRI-9439 SRI-9662

Resolution (A) 20–1.09 20–1.05No. unique reflections 79,611 86,349No. observations 289,350 312,507Mosaicity 0.7 0.55

OverallRsym (%) 4.7 5.3I/sI 9.4 9.6Data completeness (%) 99.6 97.5Mean multiplicity 3.6 3.6Wilson plot B-factor (A2) 9.8 9.4Highest shell (A) 1.15–1.09 1.09–1.05Rsym (%) 42.2 23.1I/sI 2.3 2.8Data completeness (%) 99.1 83.8Mean multiplicity 3.3 2.1

RefinementNo. reflections used in refinement 79,411 86,271Rcryst (%) 13.1 13.0Rfree (%) 17.3 18.1rms deviations, bond lengths (A) 0.013 0.017rms deviations, bond angles (deg.) 2.23 2.27Ramachandran plot (% most favored and additional allowed residues) 95.0, 5.0 93.1, 6.9No. atoms (including alternate conformations; protein, ligands, water molecules) 1627, 83, 436 1567, 82, 338Average B-factors (A2) (protein, ligands, water molecules) 18.6, 21.8, 36.8 19.5, 21.5, 39.5Data/parameter ratio 4.1 4.7

Figure 2. The structure of hDHFR is dominated by a mixed-type eight-stranded b-sheet. Stereoview of the hDHFR/NADPH/SRI-9439 ternary complex in a Ribbons46 representation. Secondary structural elements are named accordingto Freisheim & Matthews.49 NADPH and SRI-9439 are shown as stick figures (C, gray; N, blue; O, red; P, yellow-green).

Atomic Resolution Human DHFR Structures 679

of hDHFR.4 – 6 Standard nomenclature is used forthe secondary structural elements of DHFR, asshown in Figure 2. The tertiary structure of allDHFRs is dominated by a b-sheet containingseven parallel b-strands and one anti-parallelb-strand at the C terminus. We observe that thecore b-sheet is bounded by only four a-helices, incontrast to five a-helices observed in previouslypublished hDHFR crystal structures.4,5 In ourmodel, residues 102–107 form two successivetight turns rather than an a-helix; this confor-mation has been observed before,6 and is likelydue to crystal packing forces (Pro103 is in van derWaals contact with Leu153 from a symmetry-related molecule). Two cis-peptide bonds arepresent in our structures. The highly conservedcis-peptide bond (v ¼ þ4.58 [SRI-9439], þ6.28[SRI-9662]) between residues Gly116 and Gly117has been observed in all DHFR crystal structuresto date. A cis-peptide bond (v ¼ 25.48, 29.28) ispresent between Arg65 and Pro66; this cis-peptidebond has been observed in several other DHFRcrystal structures,5,7 – 9 but not in one earlierhDHFR structure.4

The 5-deazapteridine rings of SRI-9439 andSRI-9662 bind identically to the DHFRactive site

Positive electron density for both SRI-9439 andthe pteridine ring of SRI-9662 became apparent in2FO 2 FC and FO 2 FC Fourier maps early in refine-ment, facilitating the unambiguous placement ofthe inhibitors in the folate binding site (Figure 3).As originally observed for methotrexate,10 and sub-sequently other DHFR inhibitors, both inhibitorsbind with their 5-deazapteridine rings flippedapproximately 1808 along the ring long axisrelative to the position of folate in the active site.Thus, the opposite side of the pteridine ring ispresented to the NADPH cofactor.

The inhibitors bind in a hydrophobic pocketadjacent to helix aB, with the 5-deazapteridinering almost perpendicular to the 5-quinolylaminogroup (SRI-9439) or the 2,5-dimethoxyphenylgroup (SRI-9662). The 5-deazapteridine ring of theinhibitors forms hydrophobic contacts with Val8and (for SRI-9662) weakly with Leu22 (Figure 4).These conserved residues form the floor of the

Figure 3. The inhibitors bind in the hDHFR active site in an unambiguous conformation. In this stereoview, rotated,1808 from the orientation of Figure 1, (a) SRI-9439 and (b) SRI-9662 are shown as found in the hDHFR/NADPHternary complexes. The 1.5 A resolution FO 2 FC electron density maps, contoured at 2.5 s, show the positive electrondensity in the active site prior to the addition of either inhibitor to the molecular models. Ligands are colored as before;the protein C atoms are sea-green. Selected hydrogen bonds are shown by lines (broken lines if .3.0 A). Noteespecially the hydrogen bonds between the 5-deazapteridine ring and Glu30, and the distinct water-mediatedhydrogen bonds to the lipophilic moieties.

680 Atomic Resolution Human DHFR Structures

Figure 4. The 5-deazapteridine rings of both inhibitors form identical contacts in the active site. LIGPLOT50 diagramsof the hydrogen bonding and van der Waals interactions of SRI-9439 (left) and SRI-9662 (right) in the hDHFR activesite. The aromatic rings of both inhibitors form similar hydrophobic interactions in the folate-binding site, but thehydrogen-bond mediated interactions are distinct.

Figure 5. The 5-deazapteridinerings of both inhibitors puckerdramatically upon binding tohDHFR. (a) The bound confor-mations of both SRI-9439 (C, gray;N, blue) and SRI-9662 (C, steel-blue;N, cyan) exhibit a pronounced puck-ering in the 5-deazapteridine ring,which allows optimal interactionswith hDHFR. Despite the confor-mational constraint of the cis doublebond, SRI-9662 occupies a similarspatial region as SRI-9439. (b) The5-deazapteridine ring of SRI-9439(and SRI-9662) is much more puck-ered than the deazapteridine ringsof unbound trimetrexate (TMQ; C,gold; N, cyan) or piritrexim (PTX;C, tan; N, cyan). (c) PTX bound tohDHFR (Leu22Phe mutant51) iscanted in the active site comparedto SRI-9439.

Atomic Resolution Human DHFR Structures 681

hydrophobic pocket to which methotrexate andother inhibitors bind.6,10 As with folate binding,Phe34 in helix aB forms hydrophobic interactionswith the face of the 5-deazapteridine ring oppositethat of Leu22.4 The 5-deazapteridine rings of bothinhibitors therefore bind to the hDHFR active sitein an identical fashion, as do other inhibitors ofhDHFR. The average temperature factors for the5-deazapteridine rings of SRI-9439 and SRI-9662 inthe ternary complexes are similar (11.2 A2 and12.5 A2, respectively), whereas the temperaturefactors of the 5-quinolylamino and the 2,5-dimethoxyphenyl groups of SRI-9439 and SRI-9662 are significantly different (15.6 A2 and27.0 A2, respectively). The similarities in the bind-ing geometry and degree of order (temperaturefactors) of the 5-deazapteridine rings of theseinhibitors, coupled with the differences observedfor their lipophilic moieties, suggests that theobserved differences in binding affinity are modu-lated by these lipophilic side-chains. This notion isin accord with extensive enzymatic test data.3

Tight hydrogen bonds link the 5-deazapteridine ring to DHFR

The N1 and N2 nitrogen atoms of the 5-deazap-teridine rings form hydrogen bonds to oxygenatoms O12 and O11 of the side-chain of Glu30 withdistances of 2.76 A and 2.78 A (SRI-9439) and2.78/2.77 A (SRI-9662) (Figure 3). Glu30, which ishighly conserved in the active site of all vertebralDHFRs, is replaced by aspartate in T. gondiiDHFR-TS and in bacterial DHFRs.7 The carboxylategroup of Glu30 is essentially coplanar with the5-deazapteridine rings of SRI-9662 and SRI-9439.The angle between the two planes is 5.28 and 8.28

for the SRI-9439 and SRI-9662 ternary complexes,respectively.

In both ternary complexes, Glu30 participates ina “tightly constrained polar pocket” that has beenobserved in all other DHFR structures determinedto date.7,9,11 The O11 atom of Glu30 forms a hydro-gen bond with the Og1 atom of Thr136 (2.71 A),which is linked in turn to two highly conservedwater molecules that are buried deep in the activesite (temperature factors 9.4/9.5 A2 and 11.3/11.7 A2 [SRI-9439/SRI-9662]). Another tightlybound water molecule (temperature factor 13.4/15.5 A2 [SRI-9439/SRI-9662]) is coordinated byatoms O12 of Glu30 (2.81 A), N11 of Trp24 (3.09 A),N8 of the 5-deazapteridine ring (2.99 A), andanother water molecule buried in the active site(2.76 A; SRI-9439 only, though weak differenceelectron density in the SRI-9662 complex suggeststhat this water may be present at partialoccupancy).

Puckered 5-deazapteridine rings of SRI-9439and SRI-9662 orient the exocyclic atomsfor binding

The DHFR-bound 5-deazapteridine rings of SRI-9439 and SRI-9662 are essentially identical in bothstructure and geometry. As shown in Figure 5,both exhibit a pronounced pucker, with the C4and C5 atoms out of the plane of the ring in oppo-site directions by as much as 0.2 A (Table 2). Thispucker forces the exocyclic amino nitrogen N4atom (bonded to C4) of both inhibitors towardsthe nicotinamide ring of NADPH, positioning it toform hydrogen bonds with the backbone carbonyloxygen atoms of Ile7 (2.89/2.95 A [SRI-9439/SRI-9662]) and Val115 (2.96/2.97 A) (Figure 4). The exo-cyclic methyl carbon atom C5A (bonded to C5) is

Table 2. Geometrical distortion of the 5-deazapteridine rings of SRI-9439 and SRI-9662

Deviation from best plane(A)b

InhibitorPlanarity rmsd

(A)a C4 C5 N4 C5AN4–C5A distance

(A)N4–C4–C5–C5A pseudotorsion anglec

(deg.)

SRI-9439 0.069 20.18 0.13 20.43 0.44 3.0 20SRI-9662 0.044 20.10 0.20 20.23 0.58 3.0 19PTXc 0.023 20.056 0.049 20.135 0.112 2.9 5TMQ-1c 0.014 20.050 0.053 20.146 0.147 2.9 7TMQ-2c 0.068 20.199 0.031 20.459 0.168 2.9 16CCDC searchd

Average 0.028 20.077 0.047 20.218 0.200 N/A N/AMin 0.010 20.161 20.019 20.383 20.087Max 0.045 20.024 0.079 0.037 0.367Sd 0.010 0.042 0.026 0.108 0.128

a Best plane defined by 5-deazapteridine (5,8-dideazapteridine for TMQ) ring system atoms: N1, C2, N3, C4A, C6, C7, N8 (C8).b Negative deviations are toward the nicotinamide ring of NADPH; positive deviations are toward the benzene ring of Phe34.c PTX coordinates taken from Sutton & Cody16 (CCDC entry GIBHUU); TMQ-1 coordinates taken from Sutton & Cody17 (CCDC

entry FOGWAZ); and TMQ-2 coordinates taken from Hempel et al.18 (CCDC entry FUFQUS; molecule 2).d CCDC entries BAZWUU, BEBCIU (both molecules), BEBCIU01, BEBFIX, BIQCEJ, BIQCUZ, BUMHUM, HAYQON, HAYQUT,

KEKZIJ, LIYNOW, MMANCX, MONPHB (both molecules), NANMEK, NANPCX, NANPCX, and VIGJEA. GIBHUU, FOGWAZ,and FUFQUS was omitted from this composite analysis, as were ZESFAE, ZESFEI, and ZESFIM (all similar fused pentacyclicmolecules wherein the saturated center ring significantly distorts the entire molecule).

682 Atomic Resolution Human DHFR Structures

forced out of the plane of the 5-deazapteridine ringin the opposite direction, where it forms hydro-phobic contacts with residues Val115 and Phe34(Figure 4). The van der Waals contact distancesbetween C5A and the side-chains of Val115 andPhe34 are 4.1/4.3 A and 3.8/4.2 A, respectively.The positional estimated standard deviations forN4 and C5A are ,0.02 A and ,0.05 A, based ontheir temperature factors (9.5/9.5 A2 and 11.9/15.9 A2), which are much smaller than the observedout-of-plane character (as much as 0.58 A) of theseatoms (Table 2). This pronounced puckering wasconfirmed by unsuccessful refinement withplanarity restraints (see Materials and Methods).

Atomic-resolution crystal structures of metho-trexate are observed to have exocyclic atoms thatare coplanar with the pteridine ring.12,13 Inaddition, 1.6 A structures of Escherichia coli DHFRwith bound folate and Candida albicans DHFR withbound 1,3-diamino-7-(1-ethylpropyl)-7H-pyrrolo-[3,2-f ]quinazoline (PDB accession numbers 1RA2and 1AOE) were observed to have exocyclic con-stituents that were coplanar with the pteridinering.14,15 It was therefore surprising to observe inthe ternary complexes presented here that the N4and C5A atoms of both SRI-9439 and SRI-9662 aredisplaced substantially out of the plane of the5-deazapteridine ring. As shown by a lack ofresidual difference electron density around theinhibitors as well as by their thermal ellipsoid

plots (Figure 6; discussed in more detail inMaterials and Methods), it is clear that the substan-tial puckering of SRI-9439 and SRI-9662 is not dueto a superposition of multiple bound confor-mations, but is instead due to actual bending ofthe inhibitors. For example, a composite modelwould exhibit displacement of certain atoms (e.g.N8, which is diametrically opposed to C5 andC5A) to other positions (dependent on the exactlocation of the center of gyration), which would berevealed by either residual difference electron den-sity or by exaggerated thermal ellipsoids in theappropriate direction.

A search of the Cambridge CrystallographicData Centre database (version 5.22 searched withthe Conquest software supplied by CCDC) forcrystal structures of small molecules containing an(aza)napthalene ring system with exocyclicnitrogen and carbon atoms attached to C4 and C5(pteridine numbering) yielded ,20 structures. Asshown in Table 2, all of these atomic-resolutionstructures exhibited puckering of the C4 and C5atoms (as well as the exocyclic substituents) quali-tatively similar, though of lower magnitude, tothat observed here. The C4 and C5 carbon atomslie out of the plane defined by the other eightatoms of the (aza)napthalene ring by a small dis-tance, usually less than 0.1 A, while the exocyclicnitrogen and carbon atoms are puckered out ofthe plane by more substantial (average 0.21 A)

Figure 6. Thermal ellipsoid plot of SRI-9439 as bound to hDHFR. The semi-transparent thermal ellipsoids are con-toured at the 50% probability level, and the principal vibration axes are shown as sticks in this stereoview. The ellip-soids are colored according to the isotropic temperature factor, from blue to red (8–17 A2). SRI-9439 is shown as astick model colored by atom type. Top: SRI-9439. Middle: Rotated by 908 about the vertical. Bottom: Further rotatedby 908 about the horizontal. The Figure was generated with RASTEP and Raster3D.48

Atomic Resolution Human DHFR Structures 683

amounts (Table 2). Both deviations are, however,less than that observed here for the DHFR-boundinhibitors.

Of the small molecules analyzed, piritrexim(PTX) and trimetrexate (TMQ) are the most similarin structure to SRI-9439 and SRI-9662 (Figure 1).PTX differs from SRI-9662 only in that it has asingle carbon atom, rather than two, connectingthe pteridine and dimethoxybenzene rings; TMQis an 8-deaza analogue (quinazoline ring) with aCH2NH linker to a 3,4,5-trimethoxybenzene ring.The crystal structures of PTX16 and TMQ17 revealpuckering of the C4 and C5 atoms of the deazap-teridine rings as well as their exocyclic N4 andC5A substituents. Yet, as shown in Figure 5, N4and C5A of these inhibitors lie out of the 5-deazap-teridine (quinazoline) plane by only 0.11–0.14 A(Table 2), significantly smaller distances than thoseobserved for DHFR-bound SRI-9439 and SRI-9662(0.23–0.58 A). In a recently reported TMQ crystalstructure, the four TMQ molecules in the asym-metric unit exhibited varying degrees ofpuckering.18 The most puckered moleculeapproaches the level of distortion shown by SRI-9439 and SRI-9662 at C4 and N4, but not at C5and C5A, apparently due to a very tight hydro-gen-bonding network (Table 2). Two othermeasures illustrate the exaggerated puckering ofthe DHFR-bound inhibitors. First, both the PTXand the TMQ N4 and C5A atoms are separated by2.9 A, whereas the increased puckering of the5-deazapteridine rings of SRI-9439 and SRI-9662places N4 and C5A 3.0 A apart. Second, thepseudotorsion angle defined by atoms N4–C4–C5–C5A is 58 for PTX and 78 (or 168) for TMQ,whereas it increases to 208 for SRI-9439 and 198 forSRI-9662.

Given that the DHFR-bound inhibitors are sig-nificantly more puckered than are isolated PTXand TMQ, most of this increased puckering mustbe due to the formation of favorable contactsbetween the SRI inhibitors and the amino acid resi-dues of the DHFR active site. This conclusion issupported by semi-empirical theoretical calcu-lations at the PM3 level of theory.19 The calcu-lations indicated that puckering of PTX and TMQ,as seen in their small molecule crystal structures,is actually slightly more favorable than the flatstructures, by a few tenths of kcal mol21

(1 cal ¼ 4.184 J). Further puckering, of a magnitudeobserved here for SRI-9439 and SRI-9662 whenbound to hDHFR, is achieved easily, and raisesthe energy by less than 0.5 kcal mol21.

The torsionally constrained cis double bond inSRI-9662 does not cause thedimethoxybenzene ring to adopt an orientationdifferent from that of the quinoline ring of SRI-9439

Given the presence of a cis double bond in thelinker region of SRI-9662, it was surprising thatthis compound is a potent inhibitor of DHFR. It

was expected that the torsional constraint intro-duced by the cis double bond would interferewith the inhibitor’s ability to adopt a conformationthat would allow it to bind favorably in the activesite. In the crystal structure of the hDHFR/NADPH/SRI-9662 ternary complex, it becameclear that the dimethoxybenzene ring of SRI-9662was able to adopt an orientation similar to that ofthe quinoline ring of SRI-9439 by rotating notabout the C9/C10 double bond (C9/N10 in SRI-9439), but rather about the C10/C10 bond (Figure5). The torsion angle defined by atoms C6, C9,C10, and C10 in SRI-9662 is constrained by thedouble bond between C9 and C10 in the cis confor-mation, resulting in a value of 48. The equivalenttorsion angle (C6, C9, N10, C50) of SRI-9439 is,however, free to rotate, and it adopts a value of788. The dimethoxybenzene ring of SRI-9662 isrotated about the torsion angle defined by atomsC9, C10, C10, and C20 by 488, while the quinolinering of SRI-9439 is rotated about the equivalenttorsion angle (C9, N10, C50, C60) by 22.58. Theseobservations are in agreement with recently pub-lished crystallographic data showing that metho-trexate does not prefer to rotate about any onesingle bond in the linker region, but rather is freeto rotate about many single bonds in thisregion.12,13

The dimethoxybenzene ring of SRI-9662 there-fore accesses a similar spatial region of the DHFRactive site as the quinoline ring of SRI-9439, despitethese dramatically different torsion angles (Figure5). This point is illustrated further by the similarhydrophobic contacts that both SRI-9439 and SRI-9662 make with the side-chains of Pro61 andPhe31; the latter residue makes extended p-stack-ing interactions with the inhibitors (Figure 3). Thedistance between the centroids of the quinolinering of SRI-9439 and the benzene ring of Phe31 is4.1 A; the angle between these two planar aromaticsystems is 208. The centroid distances between thebenzene rings of SRI-9662 and Phe31 is 4.1 A;the interplanar angle is 238. Steric repulsions keepthe methoxy groups of SRI-9662 coplanar with theatoms of the benzene ring. Carbon atom C5A0

forms van der Waals contacts with the side-chainsof Phe31 and Pro61, while C2A0 forms contactswith the side-chains of residues Phe34 and Leu67as well as carbon atom C5A of the 5-deazapteri-dine ring.

Beyond non-specific van der Waals interactions,the lipophilic side-chains of the two SRI inhibitorsdiffer in their interactions with hDHFR. Thequinolylamino group of SRI-9439 forms no directhydrogen bonds with the protein, but atom N10 ofthe ring does form a tight hydrogen bond (2.80 A)to a well-ordered water molecule (temperaturefactor, 19.6 A2) that is in turn hydrogen bonded(2.66 A) to atom Od1 of Asn64 in the 310 helix(Figure 3). Atom N10 in the SRI-9439 linker regionalso participates in a tight, water-mediated hydro-gen bond to Og of Ser59 in aC; this water moleculeis also tightly hydrogen bonded to atom O20 of the

684 Atomic Resolution Human DHFR Structures

NADPH nicotinamide ribose moiety. Of the poten-tial hydrogen bond acceptors in the dimethoxy-phenyl group of SRI-9662, however, only O50

forms a weak hydrogen bond (3.09 A) with apoorly ordered (48.6 A2) water molecule that is nothydrogen bonded to the protein, but instead islinked very weakly (3.85 A) to a second watermolecule that is hydrogen bonded to Asn64 (Figure3). The O20 atom does not participate in any hydro-gen bonds with either the protein or the solvent.We believe that these distinct hydrogen bondarrangements contribute to the observed selectivitydifferences between SRI-9439 and SRI-9662 (seeConclusions).

In contrast to our findings with several otherfused ring (e.g. naphthalene) lipophilic antifolatesbound to Pneumocystis carinii DHFR (unpublishedresults), however, in which the aromatic ringinserts deep into the upper part of the active site(roughly behind Glu30 in Figure 3), in thehDHFR/NADPH/SRI-9439 ternary complex thequinoline ring points out of the active site (tothe right), towards solvent. This orientation of thequinoline ring was unexpected, and is likelyrelated to an unusual crystal contact observed inthe active site (see below).

SRI-9349 and SRI-9662 are canted in the DHFRactive site compared to piritrexim

Piritrexim adopts a distinct conformation, yetaccesses similar spatial regions of the DHFR activesite, as demonstrated in the ternary complex struc-ture of piritrexim bound to hDHFR reported byLewis et al. (PDB entry 1DLR).6 As shown in Figure5, the 5-deazapteridine ring of piritrexim is canted,compared to those of DHFR-bound SRI-9439 andSRI-9662; the interplanar dihedral angles betweenthe piritrexim deazapteridine ring and those ofSRI-9439 and SRI-9662 are 22.28 and 24.58, respect-ively. This canting is displayed also by the separ-ation of the C7 (deazapteridine ring) atoms (1.79and 1.97 A; piritrexim versus SRI-9439 and SRI-9662) and C9 (linker) atoms (1.79 A and 2.44 A),despite the near-overlap (,0.35 A) of the N1–N2region that interacts with the Glu30 carboxylategroup. The distinct conformation of piritrexim isdemonstrated further by comparison of a pseudo-torsion angle defined by C5 and C6 of thedeazapteridine ring and C10 and C20 of the lipo-philic side-chain (i.e. C5–C6–C10 –C20). In SRI-9439 and SRI-9662, these pseudotorsion angles are225.58 and 233.48, whereas the angle flips toþ51.88 in the piritrexim complex (Figure 5).Accordingly, the detailed placement of the twomethoxy groups differs between piritrexim andSRI-9662.

The piritrexim complex was determined with aDHFR mutant, Leu22Phe, wherein Phe22 impingeson the deazapteridine ring. To distinguish whetherthe distinct bound conformation of piritrexim isdue to the fact that the methylene linker is oneatom shorter, compared to the two atom linkers of

SRI-9439 and SRI-9662, or due to the active sitemutation, we compared the structure of theP. carinii DHFR/NADPH/piritrexim ternary com-plex published by Champness et al. (coordinatesnot deposited)20 and determined independently inthis laboratory (the binding mode appears to beidentical; unpublished results). Comparison withour (P. carinii ) coordinates reveals that the distinctbound conformation of piritrexim is mostly a prop-erty of the inhibitor, not the particular (mutated)form of DHFR to which it is bound. For example,the deazapteridine rings of P. carinii DHFR-boundpiritrexim and hDHFR-bound SRI-9439 and SRI-9662 remain canted (interplanar dihedral angles12.58 and 14.88), and the C7 and C9 separationsremain large (C7, 1.13 A and 1.32 A; C9, 1.04 Aand 1.70 A). The C5–C6–C10 –C20 pseudotorsionangle flips to 297.38, however, which reflects a1678 difference in the x2 torsion angle (rotation ofthe dimethoxyphenyl group around the CH2 –phenyl bond; 788) compared to hDHFR-boundpiritrexim (2898), coupled with a 468 change in x1

(CH2–deazapteridine bond; 21698 (P. carinii )versus 1458 (human)).

NADPH binding to human DHFR

The NADPH cofactor binds to hDHFR in bothternary complexes in an extended conformationthat lies across the C-terminal end of the centralb-sheet (Figure 2). In the SRI-9439 complex,NADPH was mostly well-ordered, and it could berefined with bond length and angle restraints onthe adenine ribose 20-phosphate moiety only. Inparticular, the dihydronicotinamide ring was notrestrained. By contrast, due to partial occupancy(see below), all of the NADPH atoms in the SRI-9662 complex were restrained lightly.

The puckering of the nicotinamide ribose ringsin both ternary complexes is C20-endo/C30-exo (2

3T),whereas the adenine ribose rings adopt the C20-exo/C30-endo (2

3T; SRI-9439) and C20-exo (2E; SRI-9662) puckers (Figure 7). Using the nomenclatureintroduced by Altona & Sundaralingam,21 thepseudorotation phase angles are 178.08/2179.38for the nicotinamide ribose rings (SRI-9439/SRI-9662), and 1.68/215.08 for the adenine riboserings.

Compared to the other atoms in NADPH, thosein the adenine ring are relatively disordered, likelyas a result of exposure to solvent coupled with thelack of strong, specific interactions with the proteinin this region. For example, in the SRI-9439 ternarycomplex, the adenine N7 makes a reasonablehydrogen bond with Og of Ser119 (2.57 A), but N6and N1 make only weak hydrogen bonds (2.94 Aand 3.11 A, respectively) to the poorly orderedside-chains of Glu123 and Arg77 (temperaturefactors ,30 A2 and ,70 A2). Hydrophobic inter-actions are formed between the adenine ring andthe side-chains of Leu75 and Val120. The adeninering of NADPH is more mobile than the rest ofthe cofactor (average temperature factors of

Atomic Resolution Human DHFR Structures 685

36.7 A2 and 17.6 A2, respectively, for the SRI-9439ternary complex; 31.2 A2 and 21.2 A2 for the SRI-9662 complex). This tendency for higher tempera-ture factors has been observed in some otherDHFR structures. An analysis of the temperaturefactors from seven other hDHFR ternary com-plexes in the Protein Data Bank (1BOZ, 1DLR,1DLS, 1HFP, 1HFQ, 1HFR, 1OHK) shows that themean temperature factor for the adenine ring ofthese cofactors was 38.5 A2, with a standard devi-ation of 4.0 A2. The mean temperature factor forthe cofactors, excluding adenine or adenine riboseatoms, was 29.7 A2, with a standard deviation of4.6 A2 ( p , 0.01, Student’s t-test). Similar obser-vations have been made for some E. coli DHFRstructures.8

The dihydronicotinamide group lies in ahydrophobic pocket formed by Ile16 and Leu22 aswell as the 2,5-dimethoxyphenyl (SRI-9662) or5-quinolylamino (SRI-9439) groups of the inhibitor.The oxygen and nitrogen atoms of the carboxa-mide moiety in the dihydronicotinamide groupare hydrogen bonded to the backbone nitrogen

and oxygen atoms of Ala9 at distances of 2.85 Aand 2.78 A in the SRI-9439 ternary complex(2.78 A and 2.68 A for SRI-9662) (Figure 8). The car-boxamide oxygen atom lies cis to carbon C4N ofthe dihydropyridine ring in both ternary com-plexes. In the SRI-9439 ternary complex, the atomsof the dihydropyridine ring and the carboxamidemoiety are essentially coplanar; the six ring atomsdeviate from a plane by rms 0.010 A, and thisplane make an angle of just 4.78 with the carboxa-mide plane. Similar coplanar geometry has beenobserved in other DHFR structures as well as inthe atomic-resolution structures of other NADPHoxidoreductases.22,23 Demonstrating the reducednature of the dihydropyridine ring, the (unrest-rained) bond lengths around the ring (N1N–C2N,C2N–C3N,…,C6N–N1N) are 1.40, 1.36, 1.52, 1.55,1.32, 1.43 A, and the C3N–C4N–C5N bond angle(1098) further demonstrates that C4N is sp3

hybridized. These alternating bond lengths are dis-tinct from the regularity observed in the 1.15 Aresolution structure of NADPþ bound to humanbiliverdin IXb reductase, reported by Pereira

Figure 8. DMSO is present at partial occupancy in the active site of the SRI-9662 ternary complex. Two orthogonalviews of the binding of NADPH in the SRI-9662 ternary complex are shown. The slight puckering of the NADPHC4N atom out of the plane of the dihydropyridine ring is apparent. The sulfur atom of a DMSO molecule, present at30% occupancy, sits 1.0 A above the C7N atom of the carboxamide moiety, while the oxygen atom of DMSO replacesthe oxygen atom of the carboxamide. The 1.05 A resolution 2FO 2 FC electron density map (purple) is contoured at2.5 s, and the 1.05 A resolution FO 2 FC electron density map prior to inclusion of DMSO in the model is contouredat 5 s (cyan) and 2.5 s (yellow-green).

Figure 7. The nicotinamide ribose ring of NADPH adopts the 23T conformation. NADPH bound in the SRI-9439 tern-

ary complex is shown as a ball-and-stick figure. The 1.09 A resolution 2FO 2 FC electron density map is contoured at 2s (cyan) and 1 s (purple).

686 Atomic Resolution Human DHFR Structures

et al.,24 in which the pyridinium ring bond lengthsaverage 1.40 A (range: 1.37–1.44 A). Whereas N1Nand C4N lie only 0.016 A and 0.008 A from thebest plane, the ribose carbon atom to which thedihydropyridine ring is bonded, C1R, lies out ofthe plane by 20.32 A (i.e. N1N is also sp3

hybridized). C4N is located 4.9 A from C7 and4.3 A from C6 in the 5-deazapteridine ring (thesites of reduction in folate and dihydrofolate,respectively). In summary, in the SRI-9439 ternarycomplex, NADPH is clearly in the reduced form,yet the dihydropyridine ring is not puckered. Theessentially flat geometry of NADPH observed hereis distinct from the strongly puckered hydroxideadduct of NADH bound to horse liver alcoholdehydrogenase recently reported at 1.10 Aresolution by Meijers et al.25

In the SRI-9662 ternary complex, the dihydro-nicotinamide group exhibits similar geometry.C4N appears to be slightly puckered out of theplane (rmsd 0.067 A) of the dihydropyridine ring,however, by 20.10 A, away from the inhibitor.This puckering is in the direction opposite to thatin which hydride transfer would occur in the tern-ary complex with folate, and is opposite to thatproposed in a catalytic mechanism for DHFR.8 Incontrast to the reliable results obtained with SRI-9439, the apparent puckering of NADPH in theSRI-9662 ternary complex should be viewed withcaution, because NADPH appears to be bound atonly ,70% occupancy.

In the SRI-9662 ternary complex, DMSO (fromthe inhibitor stock solution, and present in thecrystallization mixture at ,0.35 M) occupies thenicotinamide group’s position in the NADPHbinding site at ,30% occupancy; NADPH is thus

modeled at ,70% occupancy. The sulfur atom ofDMSO sits 1.0 A above the site occupied by theC7N carbon atom of the amide moiety (Figure 8).The oxygen atom of DMSO replaces the amideoxygen atom of the cofactor, forming a hydrogenbond (2.91 A) with the backbone nitrogen atom ofAla9. The reasons for the differences observedbetween NADPH binding in the SRI-9439 andSRI-9662 complexes are not clear.

A crystal contact mimics the binding ofclassical (anti)folates to hDHFR

In crystal structures of DHFR/NADPH/metho-trexate ternary complexes, the a-carboxylategroup of the inhibitor forms a salt-bridge with theguanidinium ion of a highly conserved arginineresidue (Arg70 in hDHFR; Arg57 in Lactobacilluscasei and E. coli DHFRs).10 This key interaction hasbeen observed in other folate and “classical”, gluta-mate-containing antifolate DHFR complexes.4,6,7,11

Such an interaction is not observed betweenDHFR and “non-classical” lipophilic antifolates,because they lack the glutamate a-carboxylategroup. In both the ternary complexes presentedhere, however, a novel salt-bridge is observedbetween the side-chain of Glu44, from a sym-metry-related DHFR molecule in the crystal lattice,and the guanidinium group of conserved Arg70.This is illustrated in Figure 9 by a least-squaressuperposition of the hDHFR/NADPH/metho-trexate ternary complex (PDB entry 1DLS)6 ontothe hDHFR/NADPH/SRI-9439 ternary complex.Thus, the side-chain carboxylate group of symm-Glu44 takes the place of the a-carboxylate groupof folate and classical antifolates like methotrexate,

Figure 9. A crystal packing interaction mimics the binding of classical antifolates to DHFR. The side chain of Glu44belonging to a hDHFR molecule related by crystallographic symmetry is bound deeply in the hDHFR active site.This stereoview illustrates a least-squares superposition of the hDHFR/NADPH/methotrexate6 and hDHFR/NADPH/SRI-9439 ternary complexes, superimposed on the solvent-accessible molecular surface of the SRI-9439 tern-ary complex. The symm-Glu44 carboxylate group (C, gold) in the SRI-9439 (C, grey) ternary complex adopts the sameposition as the a-carboxylate group of methotrexate (C, magenta), allowing it to form a double salt-bridge with theside-chain of Arg70 (C, sea-green) in the upper part of the active-site cleft. A part of the Ca trace of the symmetry-related molecule is shown. Part of NADPH is visible at the lower right. The molecular surface, colored by electrostaticpotential (red, 240 kT; white, neutral; blue, þ40 kT), was calculated with a 1.4 A radius probe using GRASP.47

Residues Arg28, Lys63, and Arg70 were omitted from the surface calculation for clarity. The Figure was preparedusing Ribbons46 and rendered with POV4GRASP (Nicolas Calimet, University of Strasbourg, France, http://pov4grasp.free.fr

Atomic Resolution Human DHFR Structures 687

an interaction that plays a key role in the recog-nition of these compounds in the DHFR activesite. (The novel and intimate active-siteinteractions observed here bear no relation toligand-induced conformational changes in theloop around residue 20 noted previously forP. carinii DHFR26 and E. coli DHFR.14 Indeed,whereas the conformation of the helical turn bear-ing Glu44 is essentially identical with thatobserved previously for the hDHFR/NADPH/piritrexim or methotrexate complexes,6 the crystalpacking in this region is very different.) Asexpected, the side-chains of some residues (e.g.Gln35) shift to accommodate insertion of the side-chain carboxylate group of symm-Glu44 into theupper active site.

A key question from a drug-design perspectiveis whether this promiscuous insertion of oneDHFR molecule into another’s active site is thecause of, or is caused by, the unusual “backwards”orientation of the 5-quinoline ring of SRI-9439, aswell as the positioning of the 2,5-dimethoxy-benzene ring of SRI-9662. Clearly, simultaneousbinding of symm-Glu44 and the lipophilic side-chains of inhibitors in the upper part of the active-site cleft is not possible. Our current hypothesis isthat the inhibitors first adopt their unusual bindingmodes, which in turn, by leaving the upper activesite empty, leads to the intimate DHFR–DHFRinteraction seen here. We believe this to be thecase for the following reasons. First, SRI-9439 andSRI-9662 bind rapidly and tightly to hDHFR (asshown by normal behavior during IC50 determi-nations), the inhibitors are present in substantialmolar excess over the enzyme in the crystallizationmixture, and crystals take several weeks to form. Itseems likely therefore that the solution bindingmode of these inhibitors in the DHFR complexprior to the onset of crystallization predisposes thecomplex to crystallize in this unusual manner.Second, these crystals are grown under the sameconditions as many other hDHFR/NADPH/anti-folate complexes studied in our laboratory(unpublished results). All of these latter complexescrystallize in space groups different from theP212121 form observed here, and all lack theintimate interaction with symm-Glu44. For all ofthese other inhibitors, the lipophilic side-chainsoccupy the upper part of the active-site cleft.

Therefore, the simplest conclusion is that SRI-9439 and SRI-9662 bind to hDHFR in their unusualconformations long before crystals nucleate. Thisunusual binding mode may prevent, through aninduced subtle conformational change in theenzyme, nucleation in the crystal form that weobserve most often (C2221). In this way, nucleationof the P212121 crystal form observed here is thenable to proceed. If this scenario is correct, it impliesthat the increased polarity of the quinoline ring ofSRI-9439 relative to its naphthalene analogues, aswell as the torsionally constrained nature of SRI-9662 may be the ultimate causes of their unusualbinding motif.

Conclusions

The ternary structures presented here providedetailed structural information on how these par-ticular inhibitors bind to hDHFR. Therefore, anexplanation can be made as to the nature of thetighter binding of SRI-9349 to rat liver DHFR and,by extension, hDHFR, than SRI-9662. Since the5-deazapteridine ring of both inhibitors has thesame structure and binds in nearly identicalconformations to the active site in both ternarycomplexes, it is the lipophilic side-chain(5-quinolylamino in SRI-9439; 2,5-dimethoxy-benzene in SRI-9662) and the linker region(CH2NH versus CHvCH) that determines thestrength of binding to the enzyme. The 5-quinolyl-amino group of SRI-9439 is stabilized via water-mediated hydrogen bonds to the side-chains ofSer59 and Asn64. In addition, the hydrophobicatoms of the aromatic ring are protected fromunfavorable solvation through interactions withPhe31 and Pro61. This results in a stable confor-mation with low temperature factors (,15–20 A2).The 2,5-dimethoxybenzene ring of SRI-9662, onthe other hand, has only a single water moleculeavailable to participate in a hydrogen bond withone of its two oxygen atoms. Furthermore, thiswater molecule does not itself participate in hydro-gen bonding with the enzyme (Figure 3). Althoughthe benzene ring forms favorable hydrophobicinteractions with Phe31 and Pro61, the exposureof the methoxy groups to water results in greateranisotropy and much higher temperature factors(,40–45 A2) for these groups compared to boththe benzene ring (,23 A2) of SRI-9662 and thequinoline ring (,16 A2) of SRI-9439.

Of critical importance in designing inhibitorsthat are selective against microbial DHFRs overhDHFR is an understanding of the structural basisfor that selectivity. The two atomic-resolutionstructures of the hDHFR ternary complexes pre-sented here lend themselves to such an interpret-ation. SRI-9662 shows a marked preference forT. gondii DHFR-TS over rat liver DHFR. This selec-tivity ratio is an order of magnitude greater thanthat of SRI-9439. The decreased specificity of SRI-9439 is due to its tighter binding to rat liver and(presumably essentially identical human) DHFR.

A model of the DHFR domain of T. gondii DHFR-TS (tgDHFR) was made by aligning the sequenceof hDHFR with those of T. gondii and Leishmaniamajor DHFR-TS (lmDHFR; program ClustalW),27

superimposing the crystal structure of lmDHFR28

on the hDHFR crystal structures presented here,and mutating residues in the active site to theT. gondii sequence. Side-chains were given stan-dard rotamer conformations using O,29 guided bythe crystal structures. Through helix a1, modelcoordinates were based on the hDHFR structures,given the higher level of homology between thehuman and T. gondii sequences in this region.Because of the generally high degree of sequenceconservation between these enzymes, only two

688 Atomic Resolution Human DHFR Structures

residues that contact the inhibitor, lmDHFR Met53and Val87, differ in tgDHFR (Phe32 and Met87,which correspond to hDHFR Phe31 and Ile60).Also, the pteridine and 5-quinolylamino-coordinat-ing residues in hDHFR, Glu30 and Asn64, areaspartate and phenylalanine in both parasiteenzymes. In total, therefore, there are only fourpositions in the hDHFR and tgDHFR active siteswhere different residues (hDHFR Ile7/tgDHFRVal8, Glu30/Asp31, Ile60/Met87, and As64/Phe91) directly contact the inhibitors; theseresidues are highlighted in Figure 10. The first twochanges should affect the precise positioning of5-deazapteridine rings in the tgDHFR active site(Figure 3). The latter two changes position tgDHFRMet87 and Phe91 to make favorable hydrophobicinteractions with the lipophilic moiety of inhibi-tors. Notably, in P. carinii DHFR, a phenylalanineresidue replaces hDHFR Asn64.

We believe, therefore, that when SRI-9439 isbound to tgDHFR, Phe91 forms favorable hydro-phobic interactions with the quinoline ring. Com-pared to binding to hDHFR, however, thisinteraction comes at the expense of losing thetight, water-mediated hydrogen bonds betweenAsn64 and the quinoline nitrogen atom. Thus,SRI-9439 binds equally strongly to both hDHFRand tgDHFR. In contrast, SRI-9662 makes onlytenuous, indirect contact with Asn64 of hDHFR,mediated by two water molecules separated by3.6 A (Figure 3). When SRI-9662 binds to tgDHFR,the hydrophobic interaction it makes with Phe91is stronger, thus explaining the tenfold tighterbinding to the parasite enzyme. Mutation of Phe91to asparagine in T. gondii DHFR-TS could be usedto test this explanation.

The high thermal motion of the 2,5-dimethoxy-benzene ring of SRI-9662 is a consequence of itsdecreased binding affinity for hDHFR, an effectthat likely arises from the conformationally con-

strained cis double bond in the SRI-9662 linkerregion. Most interestingly, increasing the ability ofSRI-9662 to adopt a favorable bound conformationby providing an additional degree of torsional free-dom, that is by reduction of its cis double bond toafford the 100-fold selective inhibitor SRI-7894,paradoxically slightly improves binding to T. gondiiDHFR-TS but worsens binding (by sevenfold) tomammalian DHFRs. The origin of this worsenedbinding is not clear.

Materials and Methods

Purification and crystallization

Recombinant hDHFR was produced essentially asdescribed.30 The enzyme had a specific activity of43 mmol min21 mg21 at 37 8C, pH 7.3 (assayed accordingto Hillcoat et al.31). The protein contained a stoichiometricamount of folate, which was removed upon incubationwith NADPH followed by the lipophilic antifolate.Details will be provided elsewhere (unpublishedresults). The ternary complexes of hDHFR withNADPH and SRI-9439 or SRI-9662 were formed by mix-ing hDHFR (20 mg ml21 in 25 mM KPO4 (pH 7.0),0.1 mM EDTA, 3 mM NaN3) with 60 mM NADPH, fol-lowed 15 minutes later by 60 mM of inhibitor in DMSO(final concentrations of 2 mM NADPH and 2 mM inhibi-tor). This complex solution was mixed with an equalvolume of precipitant containing 24–33% (w/v) PEG4000, 0.2 M Li2SO4, 0.1 M Tris–HCl (pH 7.9–8.4), (and5% (v/v) glycerol for hDHFR/NADPH/SRI-9662 ternarycomplex), and equilibrated with the precipitant byhanging-drop, vapor-diffusion at 4 8C. Parallelepipedcrystals grew in spacegroup P212121 with unit cell dimen-sions a ¼ 41.47 A, b ¼ 55.28 A, c ¼ 82.80 A for the SRI-9439 complex and a ¼ 41.30 A, b ¼ 54.99 A, c ¼ 82.68 Afor the SRI-9662 complex, one molecule per asymmetricunit. Crystals were flash-cooled directly in liquid nitro-gen after harvesting into mother liquor containing 10%glycerol.

Figure 10. Hypothetical model of SRI-9439 bound to Toxoplasma gondii DHFR-TS. The model was constructed asdescribed in the text. Top: In this stereoview, the protein backbone, shown as a lavender worm, is derived from thetgDHFR model coordinates. Select residues in the active site are labeled (tgDHFR numbering). The four residues thatdiffer between hDHFR and tgDHFR are highlighted (sea-green and magenta, respectively). Bottom: Partial sequencealignment of hDHFR, tgDHFR, and lmDHFR, highlighting the four different residues.

Atomic Resolution Human DHFR Structures 689

Data collection and processing

Diffraction data were collected from the hDHFR/NADPH/SRI-9439 ternary complex on beamline 7-1 atthe Stanford Synchrotron Radiation Laboratory(l ¼ 1.08 A, MAR Research MAR 300 image plate detec-tor) at 100 K. Crystal orientation and data collectionstrategy were determined with MOSFLM.32 High-resol-ution data were collected in a single pass (110 frames,1.08, 180 seconds; paddle inserted to attenuate the inten-sity of the low-resolution data; dmax , 2.0 A), followedby a single pass for low-resolution data collection (32frames, 2.08, 15 seconds). Data were collected from thehDHFR/NADPH/SRI-9662 complex on beamline X25 atthe National Synchrotron Light Source (l ¼ 0.90 A,Brandeis B4 CCD detector) at 100 K. High-resolutiondata were collected in one pass (180 frames, 0.58, 120seconds; paddle inserted; dmax , 2.5 A), followed by asingle, low-resolution data collection pass (45 frames,2.08, 10 seconds). Diffraction data from both crystalswere integrated with MOSFLM, scaled and mergedwith SCALA,33 and placed on an absolute scale withTRUNCATE.34 Further data manipulation was per-formed with the CCP4 program suite.35 Data collectionand processing statistics are summarized in Table 1.

Structure determination and refinement

A lack of peaks in the self-rotation function inAMoRe36 suggested the presence of only a singlemolecule of hDHFR per asymmetric unit for thehDHFR/NADPH/SRI-9439 ternary complex (Vm

2.20 A3 Da21;37 45% (v/v) solvent content). Cross-rotationand translation functions (AMoRe) placed unambigu-ously an unpublished hDHFR structure previouslydetermined in our laboratory (unpublished results) inthe unit cell. Rigid-body refinement of the molecularreplacement solution resulted in a correlation coefficientof 44.9% (R-factor 43.5%; 20–4 A). Refinement inX-PLOR38 alternated with manual rebuilding in O29

resulting in an Rfree factor of 24.0% (20–1.5 A). At thispoint, SRI-9439 was added to the model in the activesite and the cis peptide bonds between residues Arg65and Pro66, and between residues Gly116 and Gly117became apparent in the 2FO 2 FC and FO 2 FC maps.Refinement proceeded with the addition of riding hydro-gen atoms and anisotropic temperature factors inREFMAC39 and ARP,40 yielding an Rfree factor of 19.4%.At this point, most of the side-chain alternate confor-mations were modeled. Anisotropic displacement par-ameters were refined in SHELXL41 and riding hydrogenatoms were added. In the final rounds of refinement,restraints for all atoms in SRI-9439 (except for a planarityrestraint on the 5-quinolylamino group) and NADPH(except for those in the adenine ring and adenineribose-2-phosphate; restraints set up as describedbelow) were removed, resulting in a final Rfree factor of17.3%. Attempts to restrain the planarity of the5-methyl-5-deazapteridine moiety of SRI-9439 resultedin large difference electron density peaks for the exo-cyclic N4 and C5A atoms, confirming their out-of-planepositions. Also, for both this structure and the SRI-9662complex (below), there was no evidence from differenceelectron density maps for residual bound folate, whichwe have observed at low occupancy in some otherhDHFR/NADPH/lipophilic antifolate complexes(unpublished results). The final model contains onemolecule of hDHFR with 185 residues, one NADPHcofactor, one SRI-9439, 436 ordered water molecules,

and two sulfate anions at full and half occupancy.Twenty-six residues were modeled with alternateconformations.

Because the two crystals were isomorphous, refine-ment of the hDHFR/NADPH/SRI-9662 ternary complexbegan by rigid-body minimization in X-PLOR using theatomic coordinates and isotropic temperature factors ofhDHFR and NADPH from the hDHFR/NADPH/SRI-9439 ternary complex, with the cis peptide bondsrestrained. Alternating between refinement in X-PLORand manual rebuilding in O resulted in an Rfree factor of23.9% (20–1.5 A). SRI-9662 was added after its densitybecame apparent in the 2FO 2 FC and FO 2 FC maps.Refinement continued with the addition of riding hydro-gen atoms and anisotropic temperature factors inREFMAC and ARP, resulting in an Rfree factor of 19.6%.Most of the side-chains with alternate conformationswere modeled at this point. Refinement was completedusing SHELXL. Refinement continued with anisotropicdisplacement parameters, addition of riding hydrogenatoms, and removal of restraints on the 5-methyl-5-dea-zapteridine moiety of SRI-9662 (bond distance andangle restraints, and a planarity restraint, were retainedon the 2,5-dimethoxybenzyl moiety of the inhibitor, butthe double bond geometry was not restrained; par-ameters were based on the crystal structure of PTX16).Weak restraints were retained on NADPH; the restraintsused for the adenine ribose-2-phosphate-5-pyro-phosphate portion were based on a 0.74 A resolutionstructure of DNA,42 the 1.10 A structure of NADHbound to horse liver alcohol dehydrogenase,25 and the1.15 A resolution structure of NADPþ bound to humanbiliverdin IXb reductase.24 Bond length restraints (noangle restraints) for the reduced nicotinamide moeitywere based on the theoretical parameters determined bySchiøtt et al.43 Several large peaks of positive differencedensity were located in the NADPH binding site, thelargest being 1.0 A away from the C7N carbon atom,and were assigned to DMSO at partial occupancy.Refinement in this manner resulted in an Rfree factorof 18.1%. The final model contains one molecule ofhDHFR, one molecule of SRI-9662, one molecule ofNADPH modeled at 70% occupancy, one moleculeof DMSO modeled at 30% occupancy, 338 ordered watermolecules, and a single sulfate anion modeled at halfoccupancy. Fifteen residues were modeled with alternateconformations.

Temperature factors

A note regarding temperature factors is in order. Asshown in Table 1, the average refined temperature factorfor both structures is about twice as large as that deter-mined from the experimental Wilson plot. As this resultwas unexpected, we calculated a pseudo-Wilson plot,using calculated structure factors derived from theSRI-9439 model (SHELXL output .fcf file). The pseudo-Wilson plot had a slope of 10.5 A2, in very good agree-ment with the 9.8 A2 determined from the actual experi-mental structure factors (Table 1). (SHELXL refines onlyan overall scale factor, not an overall temperature factor.)In contrast, when 10 A2 was added to or subtracted fromall atomic temperature factors and the structure factorsand pseudo-Wilson plots were recalculated, the plotshad slopes of 21.1 A2 and 1.0 A2, in disagreement withthe experimental Wilson plot. Thus, we conclude thatthe experimental Wilson plot slope is not an accurateindicator of the average value to which the atomic

690 Atomic Resolution Human DHFR Structures

temperature factors will refine. There are several likelyreasons for this behavior, including: (1) violation of theassumptions for Wilson statistics (randomly distributedatoms with equal temperature factors); and (2) thepresence of large solvent regions (George Sheldrick, per-sonal communication).

Additional experimentation using or not usingSHELXL’s bulk solvent correction (SWAT keyword)revealed that it made little difference to the values towhich the atomic temperature factors refined. However,we repeated the last stages of the refinements withoutSWAT, because the difference electron density mapswere more easily interpretable. A few rounds of refine-ment in this manner produced slightly clearer electrondensity maps and allowed for slight improvement ofthe models. This included swapping the side-chain Oand N atoms of a few Gln and Asn residues where thehydrogen-bonding pattern was not previously clear, theaddition of some more water molecules, and furtherclarification of some alternate conformations. SWAT wasreapplied in the last cycle, where it benefited the SRI-9662 structure more than the SRI-9439 structure (0.9versus 0.1% drop in the Rfree factor).

Model checking, figures and accession numbers

The quality of both models was evaluated using PRO-CHECK44 and WHATCHECK.45 Best planes and othergeometrical parameters were calculated with the CCP4program GEOMCALC. Figures were prepared with RIB-BONS,46 GRASP,47 Raster3D/RASTEP,48 and POV4-GRASP (Nicolas Calimet, Univ. of Strasbourg, France).†

Protein Data Bank accession codes

The atomic coordinates and observed structure factorshave been deposited at the RCSB Protein Data Bank withaccession numbers 1KMS (SRI-9439) and 1KMV (SRI-9662).

Acknowledgments

We are grateful to Rick Davis for assistance and DrMike Soltis (SSRL) for his help and advice in collectingthe high-resolution data from the hDHFR/NADPH/SRI-9439 complex. Professor George Sheldrick(University of Gottingen) provided advice on the use ofSHELX, and on refinement protocols. We are thankful toDr Alexandre Kuzin for helpful discussions and DrsSam Ananthan and Kent Stewart (Abbott Laboratories)for their assistance with the search of the CambridgeCrystallographic Data Centre. We would also like tothank Dr David Matthews (Pfizer, La Jolla) for supplyingus with the atomic coordinates for the Leishmania majorDHFR-TS crystal structure, and Professor SherryF. Queener (Indiana Univ. School of Medicine) for anti-folate testing. Lastly, we thank Richard Dixon (AbbottBioresearch Center) for carrying out the semi-empiricalcalculations and for helpful discussions. This work wassupported by NIH grants AI-30279 and AI-38707 (JRP).

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Edited by I. Wilson

(Received 12 December 2001; received in revised form 30 April 2002; accepted 8 May 2002)

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