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Structural Study of Piezo Channel, a Novel Eukaryotes Mechanosensitive Channel

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Structural Study of Piezo Channel, a Novel Eukaryotes Mechanosensitive Channel Structural Study of Piezo Channel, a Novel Eukaryotes Mechanosensitive Channel. Thesis by Aron Kamajaya In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 2017 (Defended July 12, 2016) ii 2017 Aron Kamajaya All Rights Reserved iii ACKNOWLEDGEMENTS First and foremost, I would like to thank Doug Rees for his support and mentorship throughout my graduate school years. I am grateful that you gave me freedom to pursue an independent and unique project, which catalyzes my academic growth to become a scientist. Over the years, you have become my role model both as a scientist and a leader. I would also express my gratitude for the members of my thesis committee: Henry Lester, Bil Clemons, and David Chan. Your insight, advice, and guidance are critical for my success in this journey. Henry, serving as TA in your class for four terms has ignited my interest in neuroscience. Bil, thank you for being such a cool and approachable person to talk to about my project outside the formal meeting. David, thank you for offering your expertise in cell biology. I love the close interaction between your lab and the Rees lab. Next, I would like to thank the members of the Rees group, former and present, for creating such an environment that is conducive for scientific research. I also would like to give a shout out to my colleagues who have been instrumental to my success. Phong Nguyen, thanks for being an awesome partner in crime and fellow ‘ESL’ student. That Costco Friday and monthly gathering really helped ease out the pressure. Thanks to Jonas Lee and Jens Kaiser for being patient and teaching me crystallography. I also would like to thank Troy Walton, Chris Gandhi, Janet Yang, Nadia Herrera, and the ‘adopted Rees group member’ Raymond Liu for your help in proofreading my lengthy manuscripts and occasionally chatting about random cool science. I would like to express my appreciation to the two individuals whom I had the pleasure to work with: Michelle Reid iv and Pavle Nikolovski. I expect to hear great things from you guys! Allen Lee, Jeffrey Lai, and Phoebe Ray, you have done a wonderful job to keep the lab running smoothly, especially in term of ordering reagents, ensuring the availability of common reagents, scheduling a timely service, resolving occasional conflict, and repair for the malfunction equipment, particularly AKTA and fermenter. I would also like to thank Grant Jensen for organizing the single particle EM sub-group, which helped me during the initial struggle to get started in my single particle EM journey. Thanks to Alasdair McDowall for aiding me with the single particle data collection and to Haoqing Wang for helping me in the EM data processing. Big thanks to Jost Vielmetter for allowing me to use the space at the Protein Expression Center (PEC) facility and providing me with a lot of useful discussion on different options to express my gigantic protein, and to Angela Ho for the assistance on developing the stable cell line. It’s been an interesting twist of fate and a thrilling journey for me to get here. Thus, I am going to mention several people who have been the heroes in my life. First, I would like to thank my wife, Monica “the bunny” Lui, who has been patiently listening to my rambling, so others don’t have to. She is the reason why I seem to be cheerful and happy at all times in the workplace. Also, I want to acknowledge my best friend and current gym buddy, Feng Chen, who’s always there with me even in my darkest hours. I want to thank Stephen Smith, my academic mentor, and Kathleen Ryan, my research advisor at UC Berkeley, for investing in my growth as a young scientist. I also want to thank the people in the Biological Technology program at Pasadena City College, Wendie Johnston and Pamela Eversole-Cire. You opened my eyes and showed me the joy of scientific research v and discoveries. I would like to thank Kathleen Talaro for believing in me when nobody did. You have been the miracle in my life, and none of these great things would have happened without you. Together with my parents-in-law, you have made the transition to a higher education possible. In addition, I would like to thank my late uncle and my auntie for bringing me to the United States and giving me a chance to succeed in America, the land of opportunity. I also thank my sister, Dina Malounda, for living the thrilling journey together with me as the generation zero in this foreign land. Last but not least, I want to thank my parents for bringing me into this world and teaching me that I can always achieve anything that I set my mind into. Thank you for investing in my education even when the finances got very tough. Thanks for all of your prayer, mom. vi ABSTRACT Piezo is a unique family of eukaryotic mechanosensitive (MS) channel. With over 2500 amino acids per subunit, intact Piezo channel is one of the largest ion channels known to date. Two versions of Piezo can be found in vertebrates, namely PIEZO1 and PIEZO2. PIEZO1 appears to play roles in processes which control physiological homeostasis, whereas PIEZO2 assumes roles in mechanical somatosensation. A number of mutations mapped onto PIEZO1 or PIEZO2 are found in several hereditary human diseases, such as Dehydrated Hereditary Stomatocytosis, Gordon syndrome, and Distal Arthrogryposis. Although biochemical and functional studies provided many insightful findings, structural study of Piezo was very minimal. Herein, I described the structural investigation of Piezo channel. In the first study, we isolated a conserved soluble domain of Piezo (C-terminal loop 2, CTL2) from the C. elegans homolog, and provided the first molecular glimpse into this enigmatic MS channel. Subsequently, I described challenges that are associated with the expression and protein preparation of the full length Piezo channel. Recently, the full length mouse PIEZO1 structure solved by single particle cryo-EM revealed trimeric arrangement of the intact channel. CTL2 domain forms an extracellular cap which makes up the central core in this Piezo model. Lastly, we isolated a stable C-terminal fragment of Piezo. This fragment corresponds to the entire central core of Piezo channel and a few upstream transmembrane helices. This fragment can be localized to the plasma membrane. Further investigation is needed to look at the functionality of this fragment. vii PUBLISHED CONTENT AND CONTRIBUTIONS Kamajaya, A., Kaiser, J.T., Lee, J., Reid, M., Rees, D.C. (2014) The structure of a conserved piezo channel domain reveals a topologically distinct β sandwich fold. Structure 22(10):1520-7. doi: 10.1016/j.str.2014.08.009. A.K. designed the experiments, performed most of the experiments, analyzed the results, and wrote the manuscript. J.T.K. and J.L. provided mentorship in x-ray diffraction data processing. M.R. performed the early protein purification. D.C.R provided the mentorship, funding, and editorial service. viii TABLE OF CONTENTS Acknowledgements…………………………………………………….......iii Abstract …………………………………………………………………..…vi Published Content and Contributions……….…………………........vii Table of Contents……………………………………...…………………viii Chapter 1: Primer on Mechanosensation and the Piezo Channel ............................................................................................................. 1 Mechanosensation ......................................................................................... 2 Bacterial Mechanosensitive (MS) Channels ................................................. 4 Mechanosensation in Multicellular Eukaryotes ........................................... 5 The Plant MSL channel Family .......................................................... 6 Diverse Families of eukaryotic MS Channels .................................... 7 Piezo Family of Metazoan MS Channel Initial Discovery .................................................................................. 9 Physiological Role ............................................................................. 10 Current Puzzles .................................................................................. 13 The Scope of the Thesis .............................................................................. 16 Chapter 1 Figure .......................................................................................... 17 References .................................................................................................... 18 ix Chapter 2: The Structure of a Conserved Piezo Channel Domain Reveals a Beta Sandwich Fold ............................................................... 33 Summary and Highlights ............................................................................. 34 Introduction .................................................................................................. 35 Results Construct Selection ............................................................................ 37 Architecture of the C. elegans CTL2 Domain .................................. 38 Structure of M31R C. elegans PIEZO CTL2 Domain Variant ........ 39 Discussion .................................................................................................... 40 Experimental Procedures ............................................................................. 42 Acknowledgements and Author Contributions .......................................... 46 References .................................................................................................... 47
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