Abstract In an effort to understand the role of specific active site residues in modulating the binding selectivity of inhibitors against dihydrofolate reductase (DHFR) from different species, a series of single and double mutants of DHFR were made at positions 35 and 64 of human (h) DHFR. To explore sequence differences between human and the pathogenic organisms Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) DHFR, the causative agent of Pneumocystis pneumonia (PcP) in patients with AIDS, kinetic data were measured for trimethoprim, used for treatment of AIDS-related pneumonia. Crystal structure data are reported for the Q35S/N64F double mutant of hDHFR as a binary complex with trimethoprim (TMP). These substitutions make the human enzyme resemble a cross between pjDHFR which has Ser/Ser at these positions and pcDHFR which has Lys/Phe at these sites. Kinetic data show enhanced affinity for the binding of TMP to this double mutant (Ki 604 nM) compared to wild type hDHFR (Ki 1757 nM). The Ki of TMP is 43 nM for pjDHFR and 222 nM for pcDHFR. Structural data for the binary complex of TMP with this double mutant of hDHFR revealed the unexpected binding of two copies of TMP; one bound in the normal binding site while the other occupies a shallow pocket different from that involved in binding the γ-glutamate of folate substrates or inhibitors. The trimethoxyphenyl ring of the second TMP makes a series of hydrophobic contacts with the trimethoxyphenyl ring of the first TMP and with the phenyl ring of residue F31 and the mutated F64 in this structure. Structural data for the binary complex of wild type hDHFR with TMP does not indicate such binding. The conformation of the two TMP in the double mutant complex differ significantly from each other and from that observed for the wild type hDHFR binary complex. Analysis of all DHFR TMP complexes reveals the bridge torsion angles fall into two broad ranges and the two TMP in the mutant hDHFR complex fall into the less populated group. These data suggest that the combination of hydrophobic contacts from the normal F31 in hDFHR and the mutant F64 provide a unique environment to create sufficient van der Waals contact energy to stabilize binding in this unusual site. Supported in part by NIH GM51670 (VC). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2676.