Dihydrofolate reductase (DHFR) is a small, ~20 kD enzyme that catalyzes the reduction of 7,8- dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF), a key metabolite required for DNA biosynthesis. Because of DHFR's central location in metabolism and required activity for cell proliferation, it has become an attractive drug target for the treatment of human cancers and infectious diseases. Furthermore, differences in sequence and structural properties in DHFRs from different organisms allow many of these drugs to act with high specificity. Under a separate light, extensive biochemical and structural studies on E. coli DHFR have led to DHFR becoming a paradigm for how dynamic fluctuations in protein structure facilitate enzyme function. DHFR is now known to undergo switching between distinct conformational states on a microsecond to millisecond timescale, in a manner that connects one step in the functional cycle to adjacent steps. Here, E. coli DHFR is studied in light of its importance as a model for both protein-drug interactions and enzyme dynamics. An interdisciplinary experimental approach combining protein NMR relaxation with transient and pre-steady-state kinetics will be employed to study the response of DHFR behavior to chemical denaturants and antifolate inhibitors with affinities spanning five orders of magnitude. Since off-rates are a key determinant of binding affinity, attention will be paid to the role of internal dynamics and conformational changes to ligand dissociation, in the cases of both natural substrates and antifolates. The role of picosecond-nanosecond fluctuations in stabilizing bound ternary states and promoting concerted conformational changes will be assessed, with particular focus on side-chain mobility. The DHFR system presents an excellent opportunity to study the influence of internal dynamics on folate/antifolate ejection from different conformational states;conversely, these studies will address how conformational context defines the dynamics of ligand occupancy and release. Given that conformational changes are collective motions, the inherent connectivity between residues will be mapped using an NMR perturbation-response approach. Throughout this research, emphasis will be placed on DHFR complexes containing reduced nicotinamide adenine dinucleotide phosphate cofactor (NADPH). In summary, this application seeks to gain mechanistic insights into the role of internal dynamics in protein-drug interactions, slow conformational changes, ligand ejection, and intramolecular communication in DHFR. Project Narrative Dihydrofolate reductase is the target for drugs used to treat cancer and infectious diseases, and it serves as a model for understanding protein-drug interactions. By using protein NMR spectroscopy and enzyme kinetics to identify mechanisms of protein flexibility that either stabilize or destabilize drug occupancy, a greater understanding of the determinants of drug binding affinity will be obtained. This new knowledge will increase the efficiency of the design of small molecule inhibitors to dihydrofolate reductase and other proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM083059-04S1
Application #
8450564
Study Section
Special Emphasis Panel (ZRG1-MSFE-S (01))
Program Officer
Anderson, Vernon
Project Start
2008-01-07
Project End
2012-12-31
Budget Start
2010-12-01
Budget End
2012-12-31
Support Year
4
Fiscal Year
2012
Total Cost
$96,029
Indirect Cost
$30,491
Name
University of North Carolina Chapel Hill
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Sapienza, Paul J; Li, Li; Williams, Tishan et al. (2016) An Ancestral Tryptophanyl-tRNA Synthetase Precursor Achieves High Catalytic Rate Enhancement without Ordered Ground-State Tertiary Structures. ACS Chem Biol 11:1661-8
Sapienza, Paul J; Lee, Andrew L (2016) Widespread Perturbation of Function, Structure, and Dynamics by a Conservative Single-Atom Substitution in Thymidylate Synthase. Biochemistry 55:5702-5713
Falk, Bradley T; Sapienza, Paul J; Lee, Andrew L (2016) Chemical shift imprint of intersubunit communication in a symmetric homodimer. Proc Natl Acad Sci U S A 113:9533-8
Sapienza, Paul J; Falk, Bradley T; Lee, Andrew L (2015) Bacterial Thymidylate Synthase Binds Two Molecules of Substrate and Cofactor without Cooperativity. J Am Chem Soc 137:14260-3
Sapienza, Paul J; Lee, Andrew L (2014) Backbone and ILV methyl resonance assignments of E. coli thymidylate synthase bound to cofactor and a nucleotide analogue. Biomol NMR Assign 8:195-9
Wang, Zhen; Sapienza, Paul J; Abeysinghe, Thelma et al. (2013) Mg2+ binds to the surface of thymidylate synthase and affects hydride transfer at the interior active site. J Am Chem Soc 135:7583-92
Carroll, Mary J; Mauldin, Randall V; Gromova, Anna V et al. (2012) Evidence for dynamics in proteins as a mechanism for ligand dissociation. Nat Chem Biol 8:246-52
Mauldin, Randall V; Sapienza, Paul J; Petit, Chad M et al. (2012) Structure and dynamics of the G121V dihydrofolate reductase mutant: lessons from a transition-state inhibitor complex. PLoS One 7:e33252
Carroll, Mary J; Gromova, Anna V; Miller, Keith R et al. (2011) Direct detection of structurally resolved dynamics in a multiconformation receptor-ligand complex. J Am Chem Soc 133:6422-8
Sapienza, Paul J; Mauldin, Randall V; Lee, Andrew L (2011) Multi-timescale dynamics study of FKBP12 along the rapamycin-mTOR binding coordinate. J Mol Biol 405:378-94

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