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.
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.
|Dahlin, Jayme L; Cuellar, Matthew; Singh, Gurpreet et al. (2018) ALARM NMR for HTS triage and chemical probe validation. Curr Protoc Chem Biol 10:91-117|
|Olivieri, Cristina; Subrahmanian, Manu Veliparambil; Xia, Youlin et al. (2018) Simultaneous detection of intra- and inter-molecular paramagnetic relaxation enhancements in protein complexes. J Biomol NMR 70:133-140|
|Tomasini, Michael D; Wang, Yingjie; Karamafrooz, Adak et al. (2018) Conformational Landscape of the PRKACA-DNAJB1 Chimeric Kinase, the Driver for Fibrolamellar Hepatocellular Carcinoma. Sci Rep 8:720|
|Larsen, Erik K; Olivieri, Cristina; Walker, Caitlin et al. (2018) Probing Protein-Protein Interactions Using Asymmetric Labeling and Carbonyl-Carbon Selective Heteronuclear NMR Spectroscopy. Molecules 23:|
|Wang, Songlin; Gopinath, T; Veglia, Gianluigi (2018) Application of paramagnetic relaxation enhancements to accelerate the acquisition of 2D and 3D solid-state NMR spectra of oriented membrane proteins. Methods 138-139:54-61|
|Xia, Youlin; Rossi, Paolo; Tonelli, Marco et al. (2017) Optimization of 1H decoupling eliminates sideband artifacts in 3D TROSY-based triple resonance experiments. J Biomol NMR 69:45-52|
|Xia, Youlin; Rossi, Paolo; Subrahmanian, Manu V et al. (2017) Enhancing the sensitivity of multidimensional NMR experiments by using triply-compensated ? pulses. J Biomol NMR 69:237-243|
|Kim, Jonggul; Ahuja, Lalima G; Chao, Fa-An et al. (2017) A dynamic hydrophobic core orchestrates allostery in protein kinases. Sci Adv 3:e1600663|
|Ahuja, Lalima G; Kornev, Alexandr P; McClendon, Christopher L et al. (2017) Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer. Proc Natl Acad Sci U S A 114:E931-E940|
|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|
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