EAGER: Nature of the Pre-chemistry Ensemble in Protein Kinases

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Kinases are biological molecules that modify the rate of specific chemical reactions in cells. They and the products of these chemical reactions represent central hubs in the intracellular signaling networks in all domains of life. In addition to sub-cellular localization and temporal expression, the efficiencies of particular kinases towards specific molecular products is key in targeting input signals towards distinct physiological outputs. The goal of this project is to understand the nature of this regulation with sufficient resolution in space and time to provide the means to perturb and ultimately design signaling networks that drive specific input signals towards defined cellular responses. This project will also provide a fertile training ground for a graduate student and several undergraduate researchers in a wide variety of skills that range from computational methodology, experimental NMR and enzyme biochemistry. The undergraduates will be drawn from the pool of students who transfer from one of the seven community colleges that are part of the City University of New York (CUNY) into the City College of New York (CCNY) with a goal of encouraging them to pursue higher education in the STEM disciplines. It is notable that the graduate student who is expected on this project was formerly a transfer student who has chosen to pursue his PhD degree in Biochemistry. The transfer of a phosphate group from adenosine triphosphate (ATP) to specific serine, threonine or tyrosine residues of substrate proteins represents a central mechanism of signal transduction within cells and plays a critical role in almost all physiological processes. This transfer is enabled by biocatalysts known as protein kinases and involves the formation of a pre-chemistry complex between the kinase, the substrate and ATP. Recent studies suggest that the nature of the structural dynamics of the catalytic elements of the kinase and the phospho-acceptor region of the substrate in the context of this pre-chemistry complex determines the efficiency of the phospho-transfer reaction and contributes to its variability between specific kinase/substrate pairs. These variations in phosphorylation efficiency between kinase/substrate pairs is necessary to maintain the finely tuned network of numerous phosphorylation based signaling events occurring simultaneously in the cell. An understanding of these differences in efficiency requires the characterization in atomic detail of the conformational state/states that comprise the pre-chemistry complex. This project, using a combination of cutting-edge computational approaches, solution-state nuclear magnetic resonance (NMR) methods and biochemical assays, aims to characterize the conformational states that define the pre-chemistry complex towards the goal of addressing the following question: what determines the efficiency of phosphorylation within a kinase-substrate-ATP pre-chemistry complex? The model system chosen for this project involves the mitogen-activated protein kinase ERK2 and peptides derived from its natural substrate, the transcription factor Elk-1, that are phosphorylated with differing efficiencies.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.