title: Structural analysis of human dihydrofolate reductase as a binary complex with the potent and selective inhibitor 2,4-diamino-6-{2'-O-(3-carboxypropyl)oxydibenz[b,f]-azepin-5-yl}methylpteridine reveals an unusual binding mode.
abstract: In order to understand the structure-activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structure of the binary complex of human dihydrofolate reductase (hDHFR) with the potent and selective inhibitor 2,4-diamino-6-{2'-O-(3-carboxypropyl)oxydibenz[b,f]-azepin-5-yl}methylpteridine (PT684) was determined to 1.8 Å resolution. These data revealed that the carboxylate side chain of PT684 occupies two alternate positions, neither of which interacts with the conserved Arg70 in the active-site pocket, which in turn hydrogen bonds to water. These observations are in contrast to those reported for the ternary complex of mouse DHFR (mDHFR) with NADPH [Cody et al. (2008), Acta Cryst. D64, 977-984], in which the 3-carboxypropyl side chain of PT684 was hydrolyzed to its hydroxyl derivative, PT684a. The crystallization conditions differed for the human and mouse DHFR crystals (100 mM K2HPO4 pH 6.9, 30% ammonium sulfate for hDHFR; 15 mM Tris pH 8.3, 75 mM sodium cacodylate, PEG 4K for mDHFR). Additionally, the side chains of Phe31 and Gln35 in the hDHFR complex have a single conformation, whereas in the mDHFR complex they occupied two alternative conformations. These data show that the hDHFR complex has a decreased active-site volume compared with the mDHFR complex, as reflected in a relative shift of helix C (residues 59-64) of 1.2 Å, and a shift of 1.5 Å compared with the ternary complex of Pneumocystis carinii DHFR (pcDHFR) with the parent dibenz[b,f]azepine PT653. These data suggest that the greater inhibitory potency of PT684 against pcDHFR is consistent with the larger active-site volume of pcDHFR and the predicted interactions of the carboxylate side chain with Arg75.
authors.affiliation: Structural Biology Department, Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203, USA. cody@hwi.buffalo.edu
authors.forename: Vivian
authors.initials: V
authors.lastname: Cody
authors.forename: Jim
authors.initials: J
authors.lastname: Pace
authors.forename: Jessica
authors.initials: J
authors.lastname: Nowak
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D001552:Benzazepines / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D005493:Folic Acid Antagonists / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D011621:Pteridines / Q000737:chemistry* / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C486790:2,4-diamino-6-(2'-O-(3-carboxypropyl)oxydibenz(b,f)azepin-5-yl)methylpteridine; D001552:Benzazepines; D005493:Folic Acid Antagonists; D011621:Pteridines; D010649:Phenylalanine; D013762:Tetrahydrofolate Dehydrogenase; D000645:Ammonium Sulfate