? The role of dynamics in enzyme mechanism and allostery Enzymes are complex molecules that perform difficult chemical transformations and regulate biochemical activities that maintain cell health. Although many structures of enzymes are available, numerous aspects of enzyme function remain hidden in their dynamics and transient deformations. NMR spectroscopy, in concert with other methods, has helped to realize how dynamics assists protein function on a variety of timescales. However, such studies have been largely limited to small enzymes and proteins. Typically, enzymes are large with complex features, and they are often oligomeric and symmetric in ways that are intimately tied to their allosteric regulation/function. There is thus a need to increase access to the rich dynamics and other NMR-sensitive parameters that exist in complex enzymes and larger proteins. The work proposed here aims to 1) solve long- standing problems in the general study of allostery in symmetric homodimers and 2) gain crucial information on highly flexible regions important for the function of a (large) metabolic enzyme that is a primary target for chemotherapies. Structural and dynamic processes will be examined in the human (70 kDa) and E. coli (62 kDa) versions of thymidylate synthase (TS), which methylates deoxyuridine monophosphate (dUMP) to produce the dTMP nucleotide. TS is a symmetric homodimer that is ?half-the-sites reactive?, which is interesting from the perspective of allostery since the active sites are separated by 35 . Although the half-the-sites nature of TS gives an expectation of negative thermodynamic cooperativity between the two subunits, we have showed that substrate binding cooperativity is nonexistent or small in ecTS. By contrast, hTS has pronounced negative binding cooperativity, as well as more conformational changes and additional sequence segments of high flexibility. Comparison of residue-specific behavior in the ecTS and hTS systems will yield insights into mechanisms of allostery in symmetric homodimers. Our previous work on ecTS produced an NMR strategy that enables clean, protomer-selective observation of step-wise ligand binding that is necessary to evaluate intersubunit allosteric mechanisms in homodimers. This work will be extended to further characterize intersubunit communication in ecTS in Aim 1 and applied separately to hTS in Aim 3, along with computational work to supply molecular details of the dynamics. The knowledge gained will advance the delineation of principles of allosteric communication, which are needed to engineer or control it in proteins and improve design of allosteric drugs.
In Aim 2, structural and dynamic features of function of hTS will be determined, including the role of the highly flexible and hitherto invisible 29-residue N-terminus. In addition, the means by which a tumor-derived resistance mutation weakens affinity to cancer drug 5-FU will be investigated. In summary, this work will use a combination of NMR, ITC, crystallography, and MD simulations to reveal dynamics-based function and mechanisms of allostery in a complex, symmetric, enzyme homodimer.

Public Health Relevance

Metabolism, signal transduction, and cell behavior are under the control of enzymes, which in turn modulate their own function through conformational fluctuations and allosteric processes. This research will enhance structural biology methods for investigating homodimeric enzymes/proteins, featuring a novel method to gain protomer- selective NMR information on asymmetric states of symmetric dimers. Combined with molecular dynamics simulations, mechanisms of intersubunit allostery and functional dynamics will be evaluated in the human enzyme thymidylate synthase, which is the target for anti-cancer drugs such as 5-FU. This basic research will lead to improved engineering and design of enzymes, as well as design of allosteric drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM083059-09
Application #
9309450
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Barski, Oleg
Project Start
2008-01-07
Project End
2021-07-31
Budget Start
2017-08-01
Budget End
2018-07-31
Support Year
9
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Pharmacy
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Lee, Andrew L; Sapienza, Paul J (2018) Thermodynamic and NMR Assessment of Ligand Cooperativity and Intersubunit Communication in Symmetric Dimers: Application to Thymidylate Synthase. Front Mol Biosci 5:47
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

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