Insight into the Cargo Recognition Mechanism of Kinesin Light Chain 1 by Han Youl Lee A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Pharmacology and Toxicology University of Toronto © Copyright by Han Youl Lee 2011 Insight into the Cargo Recognition Mechanism of Kinesin Light Chain 1 Han Youl Lee Master of Science Graduate Department of Pharmacology and Toxicology University of Toronto 2011 Abstract Kinesin-1 transports various cargos along the axon, while the light chain subunits play a role in selecting the types of cargos to transport. However, the mechanisms of cargo recognition and interaction have yet to be characterized. Both c-Jun kinase-interacting protein-1 (JIP1) and alcadein-1 (ALC1) are kinesin-1 cargos and compete with each other for the axonal transport machinery. I identified two polar patches of KLC1 that play a role in the interactions with JIP1 and ALC1, respectively. The main components of these two polar patches are asparagine “clamps” surrounded by positively charged lysines. Consistent with this finding, negatively charged residues of JIP1 and ALC1 are required to interact with KLC1. By structural modeling, I narrowed down the possible key residues of KLC1 that are required for interaction with c-Jun kinase interacting protein-3 (JIP3). Together, these findings reveal the versatility of KLC in the mode of interaction with many different cargos. ii Acknowledgments First and foremost, I would like to thank my supervisor, Dr. Hee-Won Park for his dedication and understanding. I first came into the lab with very limited research experience and understanding of science. Within two years, not only did you expand my knowledge in field of science and research, but you helped me develop the tools to succeed in life. Thank you for welcoming me to the lab and giving me such a great opportunity along with your endless support for my work. I would also like to thank everyone in our lab. I’ve been lucky to have worked with such a talented group; you guys have shown me the “art of science”. I will never have been able to accomplish anything that I have done without your help. Thank you for always greeting all the good and bad times with a smile. I would like to thank Dr. McPherson and Dr. Mitchell who without any hesitation were willing to be my advisor and co-supervisor. Thank you for being a part of my graduate experience. Also, I am grateful to Dr. Pai, Dr. Salahpour, and Dr. Wells for your dedication in evaluating and greatly improving this thesis. I owe the greatest appreciation to my family and friends, who have stuck by me to this day. Thank you for your love, patience and understanding for all my life. I am honoured to call you guys my family and friends. Thank You! iii Table of Contents Acknowledgments ..................................................................................................................... iii Table of Contents ...................................................................................................................... iv List of Tables ........................................................................................................................... vii List of Figures ......................................................................................................................... viii List of Abbreviations ................................................................................................................. ix 1 Introduction ........................................................................................................................... 1 1.1 Intracellular Transport .................................................................................................... 1 1.2 Motor Proteins ................................................................................................................ 1 1.3 Kinesin ........................................................................................................................... 2 1.3.1 Kinesin Superfamily ............................................................................................ 2 1.3.2 Kinesin-1 ............................................................................................................ 3 1.3.3 Kinesin Heavy Chains ......................................................................................... 4 1.3.4 Kinesin Light Chains ........................................................................................... 6 1.4 TPR domains .................................................................................................................. 7 1.5 Introduction to Kinesin-1’s Cargo and Biological Relevance .........................................13 1.5.1 Alzheimer’s disease (AD) ..................................................................................13 1.5.2 Huntington’s Disease (HD) ................................................................................14 1.5.3 Mood Disorder ...................................................................................................15 1.5.4 Diabetes .............................................................................................................15 1.5.5 Axon Outgrowth ................................................................................................16 1.6 Clinical Relevance .........................................................................................................17 1.6.1 Kinesin Light Chain Single Nucleotide Polymorphisms .....................................17 1.6.2 Kinesin-1 as a Drug Target .................................................................................17 iv 1.7 Macromolecular Crystallography ...................................................................................18 1.8 Hypothesis & Approach .................................................................................................19 1.8.1 X-ray Crystallography ........................................................................................20 1.8.2 Biochemical & Biophysical Assays ....................................................................20 1.8.3 Analysis of Structural Model ..............................................................................21 2 Materials and Methods ..........................................................................................................23 2.1 Cloning ..........................................................................................................................23 2.2 Site-directed Mutagenesis ..............................................................................................23 2.3 Solubility Test ...............................................................................................................24 2.4 Expression .....................................................................................................................26 2.5 Purification ....................................................................................................................26 2.5.1 Cell Lysis ...........................................................................................................26 2.5.2 Metal Ion Affinity Chromatography ...................................................................27 2.5.3 Size Exclusion Chromatography .........................................................................27 2.5.4 Ion Exchange Chromatography ..........................................................................27 2.6 Binding assay using size exclusion chromatography ......................................................28 2.7 Crystallization................................................................................................................28 2.8 Isothermal Titration Calorimetry (ITC) ..........................................................................29 2.9 Structural Analysis.........................................................................................................29 3 Results ..................................................................................................................................30 3.1 Crystallography .............................................................................................................30 3.1.1 Solubility & Expression of Constructs ................................................................30 3.1.2 Purification ........................................................................................................30 3.1.3 Detection of Protein-Protein Interaction by Size Exclusion Chromatography (SEC) .................................................................................................................32 3.1.4 Crystallization ....................................................................................................33 v 3.2 Mutagenesis Binding Studies .........................................................................................34 3.2.1 Mutagenesis of KLC1-TPR ................................................................................34 3.2.2 Mutagenesis of pJIP1 and pALC1 ......................................................................39 3.3 Structural Analysis.........................................................................................................42 3.3.1 Mapping of the JIP3 and JIP4 binding site ..........................................................42 4 Discussion ............................................................................................................................43