Dissertations, Theses, and Capstone Projects

Date of Degree

9-2018

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy

Program

Biochemistry

Advisor

Ranajeet Ghose

Committee Members

Avrom J. Caplan

Kevin N. Dalby

David Jeruzalmi

J. Patrick Loria

Subject Categories

Biochemistry | Biophysics | Structural Biology

Keywords

Eukaryotic elongation factor 2 kinase

Abstract

Eukaryotic elongation factor 2 kinase (eEF-2K), the only calmodulin (CaM) dependent member of the a-kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific residue (Thr-56), decreasing its affinity for the ribosome and reducing the rate of peptide chain elongation during protein translation. In contrast to the “release-of-inhibition’ mechanism operative in most CaM-dependent proteins kinases, the activation of eEF-2K is proposed to occur through a two-step process subsequent to the engagement of CaM and involves (1) auto-phosphorylation on T348 and (2) engagement of an allosteric site by phospho-T348 leading to a state with the highest activity towards the substrate eEF-2. The precise mechanism of this CaM-mediated activation of eEF-2K and the mode of recruitment of the substrate remain poorly understood.

We utilized a variety of complimentary biophysical techniques including nuclear magnetic resonance spectroscopy (NMR), small angle X-ray scattering (SAXS), and multiple high-resolution mass spectrometric (MS) methods to determine the mechanisms through which CaM engages eEF-2K and induces conformational changes therein en routetowards its fully active state. Our integrative approach provides structural insights into the engagement of eEF-2K by CaM and represents an essential first step in defining the CaM-dependent activation of eEF-2K in atomistic detail.

Download

Share

COinS