Proteins are dynamic molecular machines, undergoing motions on a wide range of time scales. Although there is considerable evidence both from theory and experiment that many enzymes are inherently flexible, the fundamental question of how, or even if, protein fluctuations couple to catalytic function remains unanswered. Are protein motions coupled to the chemical transformation, or are they involved primarily in controlling the flux of substrate, products, or cofactors? What is the time scale of active site conformational changes required for catalysis? How is the energy landscape of the enzyme modulated during the catalytic cycle and how is it shaped during evolution? Are there species-related differences in protein dynamics and available conformational substates that might potentially be exploited for development of highly selective drugs that better discriminate between enzymes from humans and pathogens? These issues will be addressed using state-of-the-art NMR methods to elucidate the dynamic properties of an exceptionally well-characterized enzyme, dihydrofolate reductase (DHFR). DHFR is the target for anti-folate drugs such as the anticancer agent methotrexate and the antibacterials trimethoprim and iclaprim. The proposed research will focus on characterization of microsecond-millisecond time scale fluctuations in the active sites of E. coli and human DHFR, on the same time scale as key events in catalysis. NMR experiments will provide a detailed structural, dynamic, and thermodynamic description of slow motions of the active site loops in all intermediate states involved in the catalytic cycle and in carefully selected mutants that impair the catalytic process. These experiments build upon earlier research from this laboratory that showed how the energy landscape is modulated to funnel E. coli DHFR through its preferred catalytic pathway. The proposed research will provide novel insights into the energy landscapes of E. coli and human DHFR and will advance our understanding of the relationship between protein dynamics and catalytic function.

Public Health Relevance

The proposed research will address the role of protein motions in controlling the catalytic function of the enzyme dihydrofolate reductase. This enzyme is of major clinical importance as a target for anticancer agents, anti-infectives, and anti-malarial drugs. The research will provide novel information on species-related differences in protein structure and dynamics that might potentially be exploited for development of highly selective drugs that better discriminate between enzymes from humans and pathogens to decrease undesirable side effects.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM075995-08
Application #
8454460
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
2006-04-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
8
Fiscal Year
2013
Total Cost
$369,805
Indirect Cost
$175,068
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Tuttle, Lisa M; Dyson, H Jane; Wright, Peter E (2014) Side chain conformational averaging in human dihydrofolate reductase. Biochemistry 53:1134-45
Aoto, Phillip C; Fenwick, R Bryn; Kroon, Gerard J A et al. (2014) Accurate scoring of non-uniform sampling schemes for quantitative NMR. J Magn Reson 246:31-5
Fenwick, R Bryn; van den Bedem, Henry; Fraser, James S et al. (2014) Integrated description of protein dynamics from room-temperature X-ray crystallography and NMR. Proc Natl Acad Sci U S A 111:E445-54
van den Bedem, Henry; Bhabha, Gira; Yang, Kun et al. (2013) Automated identification of functional dynamic contact networks from X-ray crystallography. Nat Methods 10:896-902
Bhabha, Gira; Ekiert, Damian C; Jennewein, Madeleine et al. (2013) Divergent evolution of protein conformational dynamics in dihydrofolate reductase. Nat Struct Mol Biol 20:1243-9
Sugase, Kenji; Konuma, Tsuyoshi; Lansing, Jonathan C et al. (2013) Fast and accurate fitting of relaxation dispersion data using the flexible software package GLOVE. J Biomol NMR 56:275-83
Boehr, David D; Schnell, Jason R; McElheny, Dan et al. (2013) A distal mutation perturbs dynamic amino acid networks in dihydrofolate reductase. Biochemistry 52:4605-19
Tuttle, Lisa M; Dyson, H Jane; Wright, Peter E (2013) Side-Chain Conformational Heterogeneity of Intermediates in the Escherichia coli Dihydrofolate Reductase Catalytic Cycle. Biochemistry :
Bhabha, Gira; Tuttle, Lisa; Martinez-Yamout, Maria A et al. (2011) Identification of endogenous ligands bound to bacterially expressed human and E. coli dihydrofolate reductase by 2D NMR. FEBS Lett 585:3528-32
Bhabha, Gira; Lee, Jeeyeon; Ekiert, Damian C et al. (2011) A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis. Science 332:234-8

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