COMPARISON OF 2 INDEPENDENT CRYSTAL-STRUCTURES OF HUMAN DIHYDROFOLATE-REDUCTASE TERNARY COMPLEXES REDUCED WITH NICOTINAMIDE ADENINE-DINUCLEOTIDE PHOSPHATE AND THE VERY TIGHT-BINDING INHIBITOR PT523

Structural data for two independent crystal forms (monoclinic, C2, and orthorhombic, P2(1)2(1)2(1)) of the ternary complex of the potent ant itumor agent PT523 -deoxypteroyl)-N-delta-hemiphthaloyl-L-ornithine], reduced nicotinamide adenine dinucleotide phosphate (NADPH), and recom binant human dihydrofolate reductase (hDHFR) reveals multiple binding orientations for the hemiphthaloyl group of the inhibitor. Analysis of these data shows that PT523 binds with its pteridine ring in the same orientation observed for methotrexate (MTX) analogues, However, in ea ch structure, the hemiphthaloyl ring occupies three alternate conforma tions. In the C2 lattice, the phthaloyl moiety binds in two extended c onformations, A and C, with each conformer having a 180 degrees flip o f the o-carboxylate group, and a third, lower occupancy conformer B, w ith the phthaloyl group folded within contact of the active-site pocke t. In the orthorhombic lattice, PT523 also has three conformers for th e phthaloyl group; however, these differ from those observed in the mo noclinic lattice. Two major conformers, A and C, are displaced on eith er side of the extended position observed in the C2 lattice, one near the folded B conformer of the C2 lattice and the other extended, These conformers form tighter intermolecular contacts than those in the C2 lattice. Conformer B is folded back away from the active site in a uni que position. There are also significant differences in the conformati on of the adenine-ribose moiety of NADPH in both complexes that differ from that observed for other inhibitor-NADPH-hDHFR ternary complexes. These data suggest that the added intermolecular contacts made by the hemiphthaloyl group of PT523 contribute to its tighter binding to hDH FR than MTX, which does not extend as far from the active site and can not make these contacts. These crystallographic observations of multip le conformations for the neral agreement with solution NMR data for th e binding of PT523 to hDHFR [Johnson et al, (1997) Biochemistry 36, 43 99-4411], which show that the hemiphthaloyl group may adopt more than one conformation. However, the crystallographic data reveal more discr etely occupied positions than can be interpreted from the solution dat a. These results suggest that crystal packing interactions may influen ce their stability.