Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.

The crystal structure of the ternary complex of NADPH, the potent antifolate [2, 4-diamino-5-¿3-[3-(2-acetyloxyethyl)-3-benzyltriazen-1-yl]-4 -chloroph enyl¿-6-ethylpyrimidine] (TAB, 1) and Pneumocystis carinii dihydrofolate reductase (pcDHFR), refined to 2.1 A resolution, reveals that TAB binds similar to the antifolates trimethoprim and methotrexate. These data also reveal multiple conformations for the binding geometry of TAB with two preferred orientations of the acetyloxy and benzyl groups that results from a 180 degrees rotation about the N2-N3 triazenyl bond. The methyl of the acetyloxy and benzyl ring of TAB probes large hydrophobic regions of the p-aminobenzoyl folate binding pocket of the active site, in particular the region near Phe69, which is unique to the pcDHFR sequence. These results confirm prior molecular modeling investigations of the binding of TAB to pcDHFR that identified four low-energy binding geometries, two involving rotations about the terminal N(2)-N(3) triazenyl linkage and two involving atropisomerism about the pivotal pyrimethamine-phenyl bond. The primary differences in the molecular dynamics (MD) models and those observed in this crystal complex result from small conformational changes in active-site residues on energy minimization. However, two MD models place the acetyloxy and benzyl ring groups in a region of the active site between the cofactor-binding region and the p-aminobenzoyl folate pocket; an orientation never observed in any DHFR crystal structure to date. These conformers interact with solvent near the enzyme surface and are probably not observed due to the loss of specific hydrogen bonds with the enzyme. The high species pcDHFR selectivity of TAB could be the result of ligand flexibility that enables multiple binding orientations at the enzyme active site. Further modification of the acetyloxy region of TAB could increase its potency and selectivity for pcDHFR.