FIRST EVIDENCE OF LIGAND INDUCED CONFORMATIONAL CHANGES IN PNEUMOCYSTIS CARINII DIHYDROFOLATE REDUCTASE SUBSTRATE COMPLEXES.
V. Cody1, N. Galitsky1, D. Rak1, J.R. Luft1, W. Pangborn1 and S.F. Queener2
1Hauptman-Woodward Medical Research
Institute, Inc., 73 High St., Buffalo, New York 14203, USA
2Department of Pharmacology and
Toxicology, Indiana University, Indianapolis, IN 46202, USA.
Keywords: Dihydrofolate reductase, Pneumocystis carinii, antifolate, ligand-induced conformational changes, structure-based drug design
To treat the pneumonia caused by opportunistic infectious agents, a major cause of mortality among patients with AIDS, the design of antifolate agents with selectivity against dihydrofolate reductase (DHFR) from Pneumocystis carinii (pc) has been the focus of our structure-based design studies. As part of our program to understand the features of ligand-enzyme interactions, we have carried out the crystal structure determinations of Pneumocystis carinii dihydrofolate reductase (pcDHFR) folate (FA) binary and folate-NADP+ ternary complexes with data to 2.0 resolution for non-isomorphous crystals. The lattice of the binary FA pcDHFR complex is orthorhombic (P212121, a = 38.048 b = 61.510 c = 85.665 A) and that of the ternary FA-NADP+ pcDHFR complex is monoclinic (P21, a = 37.506 b = 43.115 c = 61.255A , b = 94.86o), similar to that observed for other pcDHFR ternary inhibitor complexes (1). These data are compared with the structure of the methotrexate (MTX) NADPH pcDHFR ternary complex which diffracts to 2.5 A in the monoclinic lattice (P21, a = 37.425 b = 43.556 c = 61.659 A, b = 94.89o ). Analysis of both folate structures revealed, for the first time, significant changes in the protein conformation, in particular near residues 23 and 47, when compared to previously reported pcDHFR structures. The change near residue 23 is the first evidence in pcDHFR of ligand induced conformational flexibility. Loop 23 (residues 21-26) is 'open' in the binary complex and 'closed' in the ternary complexes of folate and MTX, with a 6 shift in the loop. The short helical turn in loop 47 (residues 45-50) has unwound in the binary FA complex. In the unwound position, the hydrophobic residues Phe-47 and Phe-49 are exposed to the surface, whereas in the helical conformation of MTX and FA ternary complexes, Asp-46 is toward the surface. This difference changes the electrostatic potential of the enzyme surface. Data for the ternary FA pcDHFR complex also shows significant changes in the NADP+ conformation when compared to NADPH in other ternary complexes. NADP+ has a large shift in the conformation of the adenine-ribose ring of the ternary folate complex. Similar conformational changes have been reported for E. coli DHFR complexes (2), however, to date, no such changes have been observed in human DHFR structures. These results for pcDHFR could provide insight into the mechanism of ligand induced conformational changes and their affect on selectivity of binding to pcDHFR over that of human DHFR.
(Supported by GM-51670 (VC) and N01-AI-35171 (SFQ)).
1. V. Cody, N. Galitsky, J.R. Luft, W. Pangborn, A. Gangjee, R. Devraj, S.F. Queener & R.L. Blakley, Acta Cryst. D53, 638-649 (1997). 2. M. Sawaya and J. Kraut, Biochemistry, 36, 586-603 (1997).