Journal of Computer-Aided Molecular Design

, Volume 19, Issue 3, pp 165–187 | Cite as

Multiple ligand-binding modes in bacterial R67 dihydrofolate reductase

  • Hernán Alonso
  • Malcolm B. Gillies
  • Peter L. Cummins
  • Andrey A. Bliznyuk
  • Jill E. Gready


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 hydrogen-bond interactions with K32 and Y69 may play important roles.


AutoDock consensus scoring docking FlexX GROMACS interligand cooperativity ligand mobility molecular dynamics reactive complex 



R67 dihydrofolate reductase




N5- protonated dihydrofolate


reduced nicotinamide adenine dinucleotide phosphate


nicotinamide-ribose-phosphate moiety of NADPH


pterin ring of folate


nicotinamide ring of NADPH


para-aminobenzoyl glutamic acid tail of folate


adenosine diphosphate ribose moiety of NADPH


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Copyright information

© Springer 2005

Authors and Affiliations

  • Hernán Alonso
    • 1
  • Malcolm B. Gillies
    • 1
  • Peter L. Cummins
    • 1
  • Andrey A. Bliznyuk
    • 2
  • Jill E. Gready
    • 1
  1. 1.Computational Proteomics Group, John Curtin School of Medical ResearchThe Australian National UniversityCanberraAustralia
  2. 2.ANU Supercomputer FacilityThe Australian National UniversityCanberraAustralia

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