Structural Characterization of CENP-C Cupin Domains at Regional Centromeres Reveals Unique Patterns of Dimerization and Functions for the Inner Pocket by Jennifer Kui-Lok Chik A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cellular and Molecular Biology) in the University of Michigan 2019 Doctoral Committee: Assistant Professor Uhn-Soo Cho, Chair Professor Yali Dou Associate Professor Ajit P. Joglekar Associate Professor Raymond C. Trievel Jennifer Kui-Lok Chik [email protected] ORCID iD: 0000-0003-4968-5082 © Jennifer Kui-Lok Chik 2019 Acknowledgments There are a multitude of people that I must thank and acknowledge who have helped me throughout my graduate school journey. This work would not have been possible without each of these individuals, and I will always be truly grateful for their support. First and foremost, a big thank you to Uhn-Soo for allowing me to join the lab and taking a genuine interest in my development and growth as a scientist. I truly appreciate your support over the years, and I have learned so much under your guidance. I would also like to thank each of my committee members, Dr. Yali Dou, Dr. Ajit Joglekar, and Dr. Ray Trievel for your advice and insight over the years. Thank you for providing me with feedback that was always constructive and for encouraging me to think of my project from different points of view. I always left my committee meetings feeling reassured and eager to move my project forward. I must also acknowledge all the past and present members of the Cho Lab. Everyone was incredibly welcoming from day one and created a positive lab environment that was a joy to work in. Thank you for always being there for me and never hesitating to help me troubleshoot experiments or guide me in the right direction. The Cellular and Molecular Biology Program is a wonderful program with so many diverse scientific points of view, and I am incredibly proud to have been a part of it. Thank you to Dr. Bob Fuller, Dr. Kathy Collins, Cathy Mitchell, Margarita Bekiares, Pat Ocelnik, Jessica Kijek, Jim Musgrave, and Lauren Perl for helping me navigate ii through graduate school and always ensuring that everything went smoothly behind the scenes. I must also acknowledge each of the collaborators who have contributed to this work. Thank you to Dr. Lakxmi Subramanian and her lab, Ben Meinen, Dr. Phil Koldewey, and Dr. Barbara Mellone for helping me bring this cupin domain project to life. We certainly would not have been able to create this story without your time and effort. Without my undergraduate research mentors, Dr. Lori Robins and Dr. Barry Stoddard, I would not be here today. My research experiences as an undergraduate were invaluable and were instrumental in my decision to pursue graduate school. I can only hope that I will one day be able to positively impact students’ lives in the same way that you have inspired mine. I am incredibly lucky to have friends, old and new, who have never failed to be my constant pillars of support and encouragement. Thanks for never hesitating to lend an ear when I needed one most. I will always treasure the many laughs, adventures, and great meals that we shared together. Lastly, I must thank my family. There are no adequate words to describe how much you all mean to me. I am so thankful for your encouragement and unwavering support throughout this journey. Thank you for always believing that I had the potential to make it this far. I am forever grateful that I get to call you all my family. iii Table of Contents Acknowledgments......................................................................................................... ii List of Tables ................................................................................................................ ix List of Figures ............................................................................................................... x Abstract ....................................................................................................................... xiii Chapter 1: Introduction ................................................................................................. 1 Summary........................................................................................................... 1 Centromeres ..................................................................................................... 1 1.2.1 CENP-A ......................................................................................................... 2 1.2.2 Point Centromeres ........................................................................................ 4 1.2.3 Regional Centromeres .................................................................................. 5 1.2.4 Holocentromeres ........................................................................................... 6 Overview of Kinetochore Composition and Function ........................................ 6 1.3.1 The Inner Kinetochore (CCAN) ..................................................................... 7 1.3.1.1 CENP-C ................................................................................................. 7 1.3.1.2 CENP-L-N .............................................................................................. 8 1.3.1.3 CENP-T-W-S-X ...................................................................................... 9 1.3.1.4 CENP-H-I-K-M ..................................................................................... 10 1.3.1.5 CENP-O-P-Q-R-U ................................................................................ 11 iv 1.3.2 The Outer Kinetochore (KMN Network) ....................................................... 12 1.3.2.1 The Mis12 Complex ............................................................................. 12 1.3.2.2 The Knl1 Complex ................................................................................ 14 1.3.2.3 The Ndc80 Complex............................................................................. 15 1.3.3 Conservation of Kinetochore Proteins Between Model Organisms ............. 16 1.3.4 Disease Implications of Aberrant Chromosome Division and Kinetochore Dysfunction ............................................................................................................ 19 Figures ............................................................................................................ 22 Chapter 2: Characterization of Regional Centromere CENP-C Cupin Domains from S. pombe and D. melanogaster .................................................................................. 29 Introduction ..................................................................................................... 29 2.1.1 CENP-C ....................................................................................................... 29 2.1.2 Cupin Domain .............................................................................................. 32 Materials and Methods .................................................................................... 34 2.2.1 Purification of the SpCnp3CENP-C Cupin Domain .......................................... 34 2.2.2 Purification of the DmCENP-C Cupin Domain ............................................. 35 2.2.3 Crystallization and Structure Determination of the SpCnp3CENP-C Cupin Domain ................................................................................................................... 35 2.2.4 Crystallization and Structure Determination of the DmCENP-C Cupin Domain ................................................................................................................... 36 2.2.5 Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC) ................. 38 2.2.6 S. pombe Strains and Growth Assays ......................................................... 39 v Results ............................................................................................................ 40 2.3.1 Structural Determination of the S. pombe and D. melanogaster CENP-C Cupin Domains....................................................................................................... 40 2.3.2 Structural Comparison of CENP-C Cupin Domains at Point and Regional Centromeres .......................................................................................................... 41 2.3.3 Additional Secondary Structures in the CENP-C Cupin Domains from Organisms with Regional Centromeres Contribute to Dimerization and Protein Stability .................................................................................................................. 42 2.3.4 The Dimeric State of the CENP-C Cupin Domain is Evolutionarily Conserved In Vitro .................................................................................................................... 44 2.3.5 The Domain Swapped Region is Essential to Maintain the Dimeric State and Structural Integrity of the SpCnp3CENP-C Cupin Domain .......................................... 45 Discussion....................................................................................................... 46 2.4.1 Comparison of the CENP-C Cupin Domain Dimer Interface in Organisms with Point and Regional Centromeres .................................................................... 47 Author Contributions ....................................................................................... 49 Acknowledgements ........................................................................................