COMPARISON OF PNEUMOCYSTIS CARINII DIHYDROFOLATE REDUCTASE INHIBITOR-COFACTOR TERNARY COMPLEXES. Nikolai Galitsky, Vivian Cody, Joseph R. Luft, Walter Pangborn, Hauptman-Woodward Medical Research Institute, Inc., Buffalo, NY 14203; A. Gangjee, Duquesne University, Pittsburg, PA 15282, S. F. Queener, Indiana Univ., Indianapolis, IN 46202

Pneumonia caused by opportunistic infectious agents is a major cause of mortality among patients with AIDS. Antifolates have been shown effective against the dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc) which is a target for drug design studies. Crystals of recombinant Pc DHFR ternary complex with the highly selective novel furopyrimidine sulfonaphthalene antifolate are monoclinic, space group P2₁, with lattice constants of a = 37.552, b = 43.256, c = 61.389Å and [[beta]] = 94.97deg. and diffract maximally to 2.1Å resolution. Inspection of difference electron density maps of the structure refined to 2.3Å resolution with R = 18% using XPLOR revealed electron density corresponding to the cofactor NADPH and the inhibitor which was incubated with the enzyme prior to crystallization. However, the cofactor and inhibitor were refined with partial occupancy as the substrate site is also occupied by folate which remained in the enzyme after purification. The largest differences between this Pc complex and those reported previously involve the conformation of NADPH and the regions encompassing residues 58-69, 81-88 and 110-115. A twist about the pyrophosphate bond places the nicotinamide-ribose ring system further from the N4 amine of this sulfonapthyl inhibitor. There is an expansion of the active site region is this structure compared to that observed for the less selective classical furopyrimidine antifolate as reflected in the shift between the alpha atoms of S58 (1.5Å) and R82 (1.7Å). Potential energy surfaces calculated with DELPHI show that the entrance to the active site of Pc DHFR has a greater positive surface than hDHFR. Also, the only negative region on this surface is that from Glu-62 of hDHFR and Glu-63 of Pc DHFR which occupy different spatial regions on the surface. Furthermore, there is a reversal in the hydrophobicity in this region resulting from sequence changes at residues 63 and 67 (E/F and L/E, for Pc and h, respectively). These results imply that this region could have an influence on inhibitior selectivity. Supported in part by GM-51670 (VC), AI-30900 (AG), and N01-AI-35171 (SFQ).