Protein kinases are allosteric enzymes involved in cell signaling pathways (including maturation, differentiation, and metabolism) and many pathological diseases (cancer, diabetes, rheumatoid arthritis, cardiomyopathy, and others). In this proposal, we will focus on the study of allosteric binding processes of protein kinase A (PKA), a prototypical enzyme for the kinase superfamily. Although PKA has been studied for several decades, the molecular mechanisms for substrate recognition, as well as endogenous and exogenous inhibitors are still elusive. There is compelling evidence that these phenomena are mediated by the enzyme's internal dynamics. For PKA, ligand binding (nucleotides, substrates, and inhibitors) modulates the dynamic state of the enzyme, with direct repercussions on the catalytic turnover. Here, we will use a combination of high-resolution techniques (X-ray, NMR, and MD computer simulations) as well as biophysical approaches (thermocalorimetry, kinetic assays, and H/D coupled to mass spectrometry) to trace the allosteric cooperativity in PKA.
In AIM 1, we will dissect the allosteric pathways using dysfunctional mutants. In the AIM 2, we will study the dynamic activation and deactivation of the kinase by nucleotides and ATP-competitive inhibitors. Finally, in AIM 3, we will focus on the endogenous inhibition of PKI (protein kinase inhibitor) and its effects on the enzyme's internal dynamics. Understanding how allosteric signals propagate and trigger binding cooperativity will help in designing new strategies to control (by inhibiting or tuning) kinase activity for innovative therapies to treat disease.

Public Health Relevance

Kinases mediate most of signal transduction involved in metabolism, cell proliferation and differentiation, membrane transport, and apoptosis. Kinases are directly involved in pathological diseases and are preferential targets for pharmaceutical industry. Unveiling the mechanisms of allosteric signaling in kinases will provide new avenues to design novel, more selective drugs for innovating therapies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function E Study Section (MSFE)
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Gerratana, Barbara
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University of Minnesota Twin Cities
Schools of Medicine
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Kim, Jonggul; Li, Geoffrey; Walters, Michael A et al. (2016) Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A. Structure 24:353-63
Kim, Jonggul; Wang, Yingjie; Li, Geoffrey et al. (2016) A Semiautomated Assignment Protocol for Methyl Group Side Chains in Large Proteins. Methods Enzymol 566:35-57
Pagba, Cynthia V; McCaslin, Tyler G; Veglia, Gianluigi et al. (2015) A tyrosine-tryptophan dyad and radical-based charge transfer in a ribonucleotide reductase-inspired maquette. Nat Commun 6:10010
Li, Geoffrey C; Srivastava, Atul K; Kim, Jonggul et al. (2015) Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein Kinase A Using H/D Fractionation Factors. Biochemistry 54:4042-9
Kornev, Alexandr P; Taylor, Susan S (2015) Dynamics-Driven Allostery in Protein Kinases. Trends Biochem Sci 40:628-47
Chao, Fa-An; Kim, Jonggul; Xia, Youlin et al. (2014) FLAMEnGO 2.0: an enhanced fuzzy logic algorithm for structure-based assignment of methyl group resonances. J Magn Reson 245:17-23
Srivastava, Atul K; McDonald, Leanna R; Cembran, Alessandro et al. (2014) Synchronous opening and closing motions are essential for cAMP-dependent protein kinase A signaling. Structure 22:1735-43
Cembran, Alessandro; Kim, Jonggul; Gao, Jiali et al. (2014) NMR mapping of protein conformational landscapes using coordinated behavior of chemical shifts upon ligand binding. Phys Chem Chem Phys 16:6508-18
McClendon, Christopher L; Kornev, Alexandr P; Gilson, Michael K et al. (2014) Dynamic architecture of a protein kinase. Proc Natl Acad Sci U S A 111:E4623-31
Chao, Fa-An; Morelli, Aleardo; Haugner 3rd, John C et al. (2013) Structure and dynamics of a primordial catalytic fold generated by in vitro evolution. Nat Chem Biol 9:81-3

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