Journal Article10.1023/A:1014824725891
One site fits both: a model for the ternary complex of folate + NADPH in R67 dihydrofolate reductase, a D2 symmetric enzyme.
Elizabeth E. Howell,Ushma Jyotindra Shukla,Stephanie N. Hicks,R. Derike Smiley,Leslie A. Kuhn,Maria I. Zavodszky +5 more
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TL;DR: To model this ternary complex, the bound pteridine ring of folate (Fol I) from the crystal structure of R67 DHFR was used as the basis for docking the nicotinamide-ribose-Pi (NMN) moiety of NADPH, and DOCK and SLIDE, two methods for docking flexible ligands into proteins using quite different algorithms were employed.
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Abstract: R67 dihydrofolate reductase (DHFR) is a novel enzyme that confers resistance to the antibiotic trimethoprim. The crystal structure of R67 DHFR displays a toroidal structure with a central active-site pore. This homotetrameric protein exhibits 222 symmetry, with only a few residues from each chain contributing to the active site, so related sites must be used to bind both substrate (dihydrofolate) and cofactor (NADPH) in the productive R67 DHFR•NADPH•dihydrofolate complex. Whereas the site of folate binding has been partially resolved crystallographically, an interesting question remains: how can the highly symmetrical active site also bind and orient NADPH for catalysis? To model this ternary complex, we employed DOCK and SLIDE, two methods for docking flexible ligands into proteins using quite different algorithms. The bound pteridine ring of folate (Fol I) from the crystal structure of R67 DHFR was used as the basis for docking the nicotinamide-ribose-Pi (NMN) moiety of NADPH. NMN was positioned by both DOCK and SLIDE on the opposite side of the pore from Fol I, where it interacts with Fol I at the pore's center. Numerous residues serve dual roles in binding. For example, Gln 67 from both the B and D subunits has several contacts with the pteridine ring, while the same residue from the A and C subunits has several contacts with the nicotinamide ring. The residues involved in dual roles are generally amphipathic, allowing them to make both hydrophobic and hydrophilic contacts with the ligands. The result is a `hot spot' binding surface allowing the same residues to co-optimize the binding of two ligands, and orient them for catalysis.
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Citations
Importance of substrate and cofactor polarization in the active site of dihydrofolate reductase
TL;DR: An analysis of the effects of the enzyme electric field of dihydrofolate reductase on the electronic polarization of its 5-protonated dihydrosine substrate at various stages of the catalyzed hydride transfer reaction shows that the glutamate tail and the pterin ring are the highly polarized regions of the substrate.
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Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations.
TL;DR: It is illustrated that combinatorial active site mutagenesis can allow for the creation of compensatory mutations that could not be predicted and thus provides a route for more extensive exploration of functional sequence space than is allowed by point mutation.
Calorimetric studies of ligand binding in R67 dihydrofolate reductase.
Michael Jackson,Shaileja Chopra,R. Derike Smiley,Patrick O'neal Maynord,Andre Rosowsky,Robert E. London,Louis A. Levy,Thomas I. Kalman,Elizabeth E. Howell +8 more
TL;DR: A role for the O4 atom of folate in a pairing preference with NADPH, which ultimately facilitates catalysis is suggested, indicating interligand pairing preferences.
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Fragment-based design of symmetrical bis-benzimidazoles as selective inhibitors of the trimethoprim-resistant, type II R67 dihydrofolate reductase.
Dominic Bastien,Maximilian C. C. J. C. Ebert,Delphine Forge,Jacynthe L. Toulouse,Natalia Kadnikova,Florent Perron,Annie Mayence,Tien L. Huang,Jean Jacques Vanden Eynde,Joelle N. Pelletier +9 more
TL;DR: The first attempt at designing specific inhibitors to this emerging drug target by fragment-based design is reported, which procured selective inhibition of the target in the low micromolar range (K(i) = 2-4 μM).
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A balancing act between net uptake of water during dihydrofolate binding and net release of water upon NADPH binding in R67 dihydrofolate reductase.
TL;DR: The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R 67 DHFR.
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