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Structural and Functional Characterisation of Cyclin Dependent Kinase 1 Containing Complexes

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Structural and Functional Characterisation of Cyclin Dependent Kinase 1 Containing Complexes Structural and functional characterisation of cyclin dependent kinase 1 containing complexes Svitlana Mykolaivna Korolchuk Thesis submitted for the degree of Doctor of Philosophy April 2018 Northern Institute for Cancer Research Faculty of Medical Sciences Newcastle University I. Abstract CDK1 belongs to the family of serine/threonine kinases that require cyclin binding and phosphorylation for activation. CDK1 is partnered by cyclin A or cyclin B and is the only essential CDK that is required to drive the eukaryotic cell cycle. Because of their roles in driving cell growth and division, the activities of some members of the CDK family are upregulated in different cancer types making them attractive targets for therapy. The structure of monomeric CDK1 bound to CKS1 and in a complex with cyclin B are presented. These structures confirm the conserved inactive monomeric CDK fold and show how it can be remodelled by cyclin binding. These structures have revealed important insights for our understanding of CDK function and regulation as well as for the development of new more potent and specific drugs. A range of biophysical and biochemical techniques have been used to confirm and develop hypotheses as to the regulation and activity of CDK1 proposed by the structures. The melting temperature of CDK1 was estimated and compared to that of the cyclin B bound complex, CDK2 and CDK2-cyclin A to determine if the smaller interface between CDK1 and cyclin B may have implications for protein stability. The CDK1–cyclin B structures also reveal potential novel protein interaction sites that might regulate CDK1 activity. These findings inspired further investigations to explore binding sites by structural biology methods. CDK1 can phosphorylate the largest number of substrates in comparison to other CDKs. This relaxed substrate specificity was explored with reference to the insights provided by the CDK1-cyclin B-CKS2 structure. The similarities in sequence and secondary structure exhibited by the CDK family pose challenges for inhibitor design. The binding modes of a diverse set of inhibitors were characterized by a range of biophysical methods and their structures were determined by X-ray crystallography. The results of these experiments highlight the importance of conformational plasticity of the whole molecule in shaping the ATP binding site. I II. Acknowledgements This thesis would not have been possible without valuable involvement of the number of people. First of all I would like to express my deepest gratitude to my main mentor and supervisor Professor Jane Endicott who has given me a job and allowed to take it further to a next level of development and do PhD project. She has been a very knowledgeable and helpful supervisor through the years teaching me different aspects of cell cycle, molecular biology and biochemistry in general. Her endless support, encouragement and patience were vital for my progression and process of learning. I would also like to thank my second supervisor Martin Noble for his useful advice in a field of structural biology and for his optimistic approach to science and life which makes the difference. I am very grateful for both of them for reading this thesis and providing helpful feedback. I am grateful to my advisory panel Prof. Herbie Newell and Dr. Paula Salgado for their advice, expertise and guidance. The very special thanks has to be said to Nicholas Brown for introducing me to the CDK1 crystallography project and generated some constructs and initial data. This work has benefited enormously from involvement of Mat Martin and later on Daniel Wood to whom I am very grateful. Big thank you is going to Arnaud Basle for his help with crystal data collection, structure solution and his guidance. Also I want to thank my ex-colleagues Dr. Will Stanley and Dr Ruslan Moukhametzianov who participated in this project. I want to say thank you for all of the lab members for their support, advice and cheerfully stimulating atmosphere. Especially to Claire Jennings for help with different technics. Her Yorkshire steady attitude to anything she does always sets me into right direction and implements in brilliant results. I am very thankful to Richard Heath and Martina Pastok for useful discussions and advises, to Lan Wang for work she has done for this project and for everyone in a lab for their friendship and ideas. On a more personal note I would like to thank my family for all their support and patience. II III. Table of Contents I. ABSTRACT .......................................................................................................... I II. ACKNOWLEDGEMENTS .................................................................................... II III. TABLE OF CONTENTS ...................................................................................... III IV. LIST OF FIGURES .......................................................................................... VIII V. LIST OF TABLES ............................................................................................. XII VI. ABBREVIATIONS ........................................................................................... XIII INTRODUCTION .................................................................................. 1 1.1 REGULATION OF THE EUKARYOTIC CELL CYCLE ...................................................... 1 Importance of checkpoints in cell cycle regulation ...................................... 3 1.1.2 CDKs and the cell cycle .............................................................................. 4 1.1.3 Cyclins as partners that activate CDKs ....................................................... 7 1.1.4 Role of phosphorylation in CDK activation ................................................ 13 1.2 STRUCTURAL CHARACTERISATION OF THE CDK FAMILY ........................................ 14 1.2.1 A structural paradigm for CDK activation .................................................. 14 1.2.2 Structural characterisation of the cyclin family .......................................... 17 1.2.3 CDK requirements for substrate selection ................................................. 20 1.3 OTHER REGULATORS OF CDK ACTIVITY ................................................................ 23 1.4 CDKS AND CANCER ............................................................................................ 26 1.4.1 CDK4/6 and cancer ................................................................................... 27 1.4.2 CDK2 and cancer ...................................................................................... 29 1.4.3 Role of CDK9 in cancer ............................................................................. 31 1.4.4 CDK1 and cancer ...................................................................................... 32 1.4.5 Role of pharmacological CDK inhibition for cancer therapy ...................... 32 1.5 AIMS OF THE THESIS ........................................................................................... 34 STRUCTURAL INSIGHTS INTO CDK1-CONTAINING COMPLEXES .................................................................................................................................. 35 2.1 INTRODUCTION ................................................................................................... 35 Discovery of CDK1 .................................................................................... 35 Regulation of CDK1 activity ....................................................................... 38 Structures of CDK-cyclin complexes ......................................................... 40 III Structural features of cyclins A and B ....................................................... 42 Analysis of the CDK-cyclin interface ......................................................... 45 Aims and objectives .................................................................................. 47 2.2 MATERIALS AND METHODS ................................................................................. 48 Chemicals and Reagents .......................................................................... 48 Purification buffers .................................................................................... 48 Constructs used for in vitro experiments ................................................... 49 Cell culture ................................................................................................ 50 Optimisation of protein expression ............................................................ 50 Expression tests ....................................................................................... 51 Bacterial transformation and protein expression in bacterial cells ............ 51 Protein purification .................................................................................... 52 Protein purification by size-exclusion chromatography ............................. 53 SDS-PAGE ............................................................................................. 53 Preparation of complexes ....................................................................... 54 Protein crystallography ........................................................................... 54 Data collection and analysis ................................................................... 54 2.3 RESULTS AND DISCUSSION ................................................................................. 55 Purification of CDK1, cyclin B and CKS2 and complex assembly............
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