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Structural Studies on the Interaction Between Eukaryotic Elongation Factor 2 Kinase (Eef2k) and Calmodulin (Cam)

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Structural Studies on the Interaction Between Eukaryotic Elongation Factor 2 Kinase (Eef2k) and Calmodulin (Cam) University of Southampton Research Repository ePrints Soton Copyright © and Moral Rights for this thesis are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holders. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given e.g. AUTHOR (year of submission) "Full thesis title", University of Southampton, name of the University School or Department, PhD Thesis, pagination http://eprints.soton.ac.uk University of Southampton Faculty of Natural and Environmental Sciences Structural studies on the interaction between eukaryotic elongation factor 2 kinase (eEF2K) and calmodulin (CaM) Kelly Hooper Thesis for the Degree of Doctor of Philosophy June 2015 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF NATURAL AND ENVIRONMENTAL SCIENCES Biological Sciences Doctor of Philosophy Structural studies on the interaction between eukaryotic elongation factor 2 kinase (eEF2K) and calmodulin (CaM) Kelly Hooper Eukaryotic elongation factor 2 kinase (eEF2K) critically regulates translation elongation by controlling the activity of eEF2, which catalyses the translocation reaction of the ribosome. eEF2K phosphorylates eEF2 and prevents its binding to the ribosome to inhibit translation elongation. eEF2K is activated by elevated Ca2+ levels via calmodulin (CaM), although the molecular mechanism of activation is not understood. A conserved region at the N-terminus of eEF2K has been shown to be a key interaction site for CaM. A peptide corresponding to the CaM binding region of eEF2K (eEF2K82- 100) as well as longer eEF2K fragments to represent the full-length protein have been studied to dissect the molecular mechanism of eEF2K activation by Ca2+/CaM. Nuclear magnetic resonance (NMR) spectroscopy has been used to determine the binding site and the mechanism of peptide recognition and the three-dimensional solution structure of the Ca2+/CaM: eEF2K82-100 complex has been elucidated. These investigations revealed important roles for particular residues within the CaM binding region of eEF2K and in CaM itself and also resulted in a model for eEF2K activation that involves residues in the kinase domain, as well as the CaM binding region. The elucidation of this protein complex structure provides a vital tool in the study of eEF2K function, critical in cancer biology. 1 2 Table of Contents List of Figures .................................................................................................................... 9 List of Tables .................................................................................................................... 13 Academic Thesis: Declaration of Authorship ....................................................... 15 Acknowledgements ....................................................................................................... 17 Abbreviations .................................................................................................................. 19 Chapter 1. Introduction and Literature Review .................................................. 21 1.1 Motivation ....................................................................................................................... 21 1.2 Aims....................................................................................................................................... 22 1.3 Experimental strategy .................................................................................................... 22 1.4 Ca2+ signalling and calmodulin .................................................................................... 23 1.4.1 Ca2+ signalling .......................................................................................................................... 23 1.4.2 Role of calmodulin ................................................................................................................. 24 1.4.3 Structure of calmodulin ....................................................................................................... 25 1.4.4 Calmodulin target recognition ......................................................................................... 30 1.4.4.1 Calmodulin targets .......................................................................................................................... 35 1.4.4.2 Calmodulin binding motifs .......................................................................................................... 35 1.4.4.3 Structures of CaM bound to target peptides ....................................................................... 36 1.4.4.4 Structures of CaM bound to target proteins ........................................................................ 41 1.4.5 Calmodulin target regulation ............................................................................................ 42 1.5 eEF2K and translation elongation.............................................................................. 43 1.5.1 Role of eEF2K .......................................................................................................................... 43 1.5.2 Process of protein translation .......................................................................................... 44 1.5.3 Translation elongation ........................................................................................................ 45 1.5.3.1 Role of eEF2 in translation elongation ................................................................................... 46 1.5.3.2 Regulation of translation elongation ...................................................................................... 46 1.5.4 Domain structure of eEF2K ............................................................................................... 47 1.5.4.1 CaM binding region of eEF2K ..................................................................................................... 49 1.5.5 eEF2K activity ......................................................................................................................... 49 1.5.6 Regulation of eEF2K ............................................................................................................. 50 1.5.6.1 Activation of eEF2K by Ca2+/CaM ............................................................................................ 50 1.5.6.2 Regulation of eEF2K by ubiquitination and degradation .............................................. 51 1.5.6.3 Regulation of eEF2K by cell signalling pathways .............................................................. 52 1.5.6.4 Modulation of eEF2K activity by pH ....................................................................................... 53 1.5.6.5 Autophosphorylation of eEF2K ................................................................................................. 54 1.5.7 eEF2K in human disease ..................................................................................................... 55 1.6 Protein kinases ................................................................................................................. 57 1.6.1 Phosphorylation by protein kinases .............................................................................. 57 1.6.2 Evolution and classification of protein kinases ......................................................... 58 1.6.3 Protein kinase catalytic core domain ............................................................................ 61 1.6.3.1 Structure of the Protein kinase catalytic core .................................................................... 61 1.6.3.2 Catalytic mechanism of conventional ePKs ......................................................................... 62 1.6.4 Atypical protein kinases...................................................................................................... 65 1.6.5 Alpha kinases ........................................................................................................................... 65 1.6.5.1 MHCK A ................................................................................................................................................ 67 1.6.5.2 ChaK (or TRPM6 and TRPM7) ................................................................................................... 72 1.6.6 Ca2+/CaM dependent protein kinases ........................................................................... 73 1.6.6.1 Autoinhibition of Ca2+/CaM dependent protein kinases ............................................... 75 1.7 Studying protein structure and function ................................................................. 77 1.7.1 Nuclear magnetic resonance (NMR) .............................................................................. 77 1.7.1.1 Chemical shifts .................................................................................................................................. 78 3 1.7.1.2 Spin-spin coupling ........................................................................................................................... 80 1.7.1.3 Chemical exchange .........................................................................................................................
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