Date of Degree

2-2019

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Ranajeet Ghose

Committee Members

Kevin Dalby

David Jeruzalmi

Reza Khayat

Ming-Ming Zhou

Subject Categories

Biochemistry | Biophysics | Structural Biology

Keywords

calmodulin, eEF-2K, NMR, ITC, Structural biology, Biochemistry, Biophysics

Abstract

Eukaryotic elongation factor 2 kinase (eEF-2K) is a key modulator of the rate of protein synthesis. Activated by calcium-loaded calmodulin (Ca2+-CaM), eEF-2K phosphorylates its only known physiological substrate, eEF-2, on a specific threonine residue (Thr-56). Phosphorylated eEF-2 has reduced affinity for the ribosome, and results in a significant decrease in the rate of translation elongation. Modulation of the rate of translation elongation plays a crucial role in proteostasis – adequate regulation of protein synthesis, protein folding, and protein degradation that greatly influences cellular growth and survival. Binding of Ca2+-CaM triggers activation of eEF-2K and remains intact to facilitate the substrate phosphorylation, acting as a cofactor of the enzyme. Despite its importance, how the binding of Ca2+-CaM leads to activation remains poorly understood.

We employed various biophysical tools, such as nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), coupled with mutational strategies to characterize structural and thermodynamic nature of the binding of Ca2+-CaM onto eEF-2K. Our data clarify the roles of Ca2+ and each lobe of CaM that provide mechanistic insight into the structural regulation of eEF-2K mediated by the binding of Ca2+-CaM.

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