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, fundamental questions about the relationship between protein dynamics and enzyme catalysis remain unanswered. Are protein motions coupled to the chemical transformation, or are they involved primarily in controlling the flux of substrate, products, or cofactors? What role do protein motion play in progression from the preorganized ground state structure to an active site configuration that facilitates the chemical reaction? 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 and multi-conformer room temperature X-ray crystallography 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 lead to new understanding of the intrinsic molecular dynamics of this important enzyme and how its motions are modulated by interaction with substrate, cofactor, and products at various stages in the catalytic cycle. It will also provide novel insights into the role of evolution in shaping 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-malaril 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 #
2R01GM075995-09
Application #
8697848
Study Section
Macromolecular Structure and Function E Study Section (MSFE)
Program Officer
Wehrle, Janna P
Project Start
2006-04-01
Project End
2018-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
9
Fiscal Year
2014
Total Cost
$394,120
Indirect Cost
$186,141
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
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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
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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
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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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