Multiple ligand-binding modes in bacterial R67 dihydrofolate reductase Herna´n Alonso a , Malcolm B. Gillies a , Peter L. Cummins a , Andrey A. Bliznyuk b & Jill E. Gready a, * a Computational Proteomics Group, John Curtin School of Medical Research, The Australian National University, P.O. Box 334, 2601, Canberra, ACT, Australia; b ANU Supercomputer Facility, The Australian National University, 0200, Canberra, ACT, Australia Received 13 October 2004; accepted in revised form 11 March 2005 Ó Springer 2005 Key words: AutoDock, consensus scoring, docking, FlexX, GROMACS, interligand cooperativity, ligand mobility, molecular dynamics, reactive complex Summary R67 dihydrofolate reductase (DHFR), a bacterial plasmid-encoded enzyme associated with resistance to the drug trimethoprim, shows neither sequence nor structural homology with the chromosomal DHFR. It presents a highly symmetrical toroidal structure, where four identical monomers contribute to the unique central active-site pore. Two reactants (dihydrofolate, DHF), two cofactors (NADPH) or one of each (R67•DHF•NADPH) can be found simultaneously within the active site, the last one being the reactive ternary complex. As the positioning of the ligands has proven elusive to empirical determination, we addressed the problem from a theoretical perspective. Several potential structures of the ternary complex were generated using the docking programs AutoDock and FlexX. The variability among the final poses, many of which conformed to experimental data, prompted us to perform a comparative scoring analysis and molecular dynamics simulations to assess the stability of the complexes. Analysis of ligand–ligand and ligand–protein interactions along the 4 ns trajectories of eight different structures allowed us to identify important inter-ligand contacts and key protein residues. Our results, combined with published empirical data, clearly suggest that multipe binding modes of the ligands are possible within R67 DHFR. While the pterin ring of DHF and the nicotinamide ring of NADPH assume a stacked endo-conformation at the centre of the pore, probably assisted by V66, Q67 and I68, the tails of the molecules extend towards opposite ends of the cavity, adopting multiple configurations in a solvent rich-environment where hydro- gen-bond interactions with K32 and Y69 may play important roles. Abbreviations: R67 DHFR – R67 dihydrofolate reductase; DHF – dihydrofolate; DHFH+ – N5- protonated dihydrofolate; NADPH – reduced nicotinamide adenine dinucleotide phosphate; NMN – nicotinamide-ribose-phosphate moiety of NADPH; pte – pterin ring of folate; nic – nicotinamide ring of NADPH; pABA-Glu – para-aminobenzoyl glutamic acid tail of folate; 2¢,5¢-ADP – adenosine diphosphate ribose moiety of NADPH Introduction Tetrahydrofolate (THF) and its derivatives are essential cofactors derived from the vitamin folic acid. They are involved in the metabolism of the amino acids serine, glycine, and methionine and *Correspondence to: Prof Jill E. Gready, Computational Pro- teomics Group, John Curtin School of Medical Research, The Australian National University, P.O. Box 334, Canberra ACT 2601, Australia. Phone: +61 2 6125 8304, Fax: +61 2 6125 0415, Email: jill.gready@anu.edu.au Journal of Computer-Aided Molecular Design (2005) 19: 165–187 165 DOI 10.1007/s10822-005-3693-6