Syncytins and Cell Fusion in the Placenta: Structural Insights into Lipid Membrane Fusion and Placental Development by Shira Elion-Jourard A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by Shira Elion-Jourard 2018 Syncytins and Cell Fusion in the Placenta: Structural Insights into Lipid Membrane Fusion and Placental Development Shira Elion-Jourard Master of Science Department of Laboratory Medicine and Pathobiology University of Toronto 2018 Abstract Endogenous retroviruses (ERV) are genetic elements found in eukaryotes of retroviral origin. Despite making up a substantial portion of vertebrate genomes, very few ERV genes encode functional proteins. Interestingly, many animal species have evolutionarily converged in the adoption of ERV envelopes for facilitating necessary cell fusions during placental development. These placental ERV envelopes are called syncytins. Structural characterization of syncytins will provide invaluable insight into placental development and evolution as well as contribute to a general understanding of envelope facilitated membrane fusion. Crystallization of and subsequent x-ray crystallographic experiments of human syncytin-2 transmembrane ectodomain in its post-fusion conformation were carried out and the structure was solved at 1.3Å. Comparative analysis between syncytin-2 and other CX6CC type class I viral fusion proteins reveals, despite high structural conservancy, nuanced structural divergences that may reflect functional differences arising by the distinct contexts in which each protein must facilitate membrane fusion. ii Acknowledgments Joining Dr. Jeffrey Lee’s lab almost three years ago, I embarked on a journey in which I explored the world of science and research and learned more about life and myself than I ever could have imagined. I need to express my sincerest gratitude to Jeff for not only providing me the opportunity to ask scientific questions and the guidance and resources necessary to answer them but also for being an integral part of my scientific and spiritual growth. My time in the Lee lab would not have been the same without my fellow graduate students, past and present. The senior grad students in our lab: Dr. Halil Aydin, Matthew Taylor, Dr. Jonathan Cook, and Farshad Azimi welcomed me warmly and patiently taught me so much of what I know now. I would especially like to thank Dr. Aydin whose work with syncytin-1 jump started my own project and significantly contributed to this thesis. Wen-Guang He, thank you for joining our lab! You’ve brought both dry Manitoban humour and fresh scientific insight and energy to the lab. I’d also like to thank the past and present senior scientists in our lab whose guidance has been indispensable in the development of my project and my growth in science. I’d like to thank Dr. Karen Siu and Dr. Azmiri Sultana for teaching me the practical basics of working with a crystallography dataset; Dr. Peter Kojo Quashie for his guidance in protein biochemistry, and experiment design and for his mentorship throughout my degree; and Dr. Vitor Serrão, whom, unfortunately, I’ve not had much time to get to know but has clearly revitalized the lab with fresh science, and a proclivity for partying. I’d like to thank the many scientists outside of our lab who also directly or indirectly contributed to my education and experiences in the program, to name a few: my committee members, Dr. Mario Ostrowski, Dr. Jean-Phillipe Julien; as well as Dr. David Irwin, and Dr. Michael Ohh. I’d also like to thank Dr. Theo Moraes for chairing my defense. I would like to thank Rama and the other staff from the LMP graduate office for all of their help with scholarship applications and other grad school administrative tasks. I want to give a shoutout to coffee break people and Louella and other admin staff who made sure I was sociable at least one hour per week. Daniel Tarade’s rap serenades and bicycle adventures with Betty Poon will be especially memorable. Thank you for your friendship. Finally, I need to thank my family and close friends for keeping me grounded and supporting me through the inevitable ups and downs of the science rollercoaster. I would especially like to thank my roommate Jasmin Lantos for her patience with apartment cleanliness during rougher weeks and for taking me swimming that one time I really needed it. iii Table of Contents Acknowledgments.......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Abbreviations ................................................................................................................... viii Chapter 1 ..........................................................................................................................................1 Introduction to endogenous retroviruses, syncytins and viral fusion proteins ............................1 1.1 Endogenous Retroviruses.....................................................................................................1 1.1.1 Receptor Interference ...............................................................................................4 1.1.2 Immunosuppression .................................................................................................4 1.1.3 Somatic Cell Fusion .................................................................................................5 1.2 Placental Development ........................................................................................................5 1.2.1 Human and Murine Placentae ..................................................................................9 1.2.2 Syncytins in other Species .....................................................................................10 1.3 Membrane Fusion ..............................................................................................................15 1.3.1 Fusion Catalysts: The Viral Envelope ...................................................................17 1.3.2 CX6CC Subtype of Class I Fusion Proteins ...........................................................20 1.4 Summary and Rationale for Study .....................................................................................21 Chapter 2 ........................................................................................................................................23 Materials and Methods ..............................................................................................................23 2.1.1 Expression and Purification ...................................................................................23 2.1.2 Crystallization ........................................................................................................24 2.1.3 Data Collection and Processing .............................................................................25 2.1.4 Structure Determination .........................................................................................26 2.1.5 Structure Analysis .................................................................................................26 iv 2.1.6 Phylogenetic Analysis and Clustering ...................................................................27 Chapter 3 Results and Discussion ..................................................................................................28 Results and Discussion ..............................................................................................................28 3.1 Expression, Purification, and Crystallization.....................................................................28 3.2 Structure Determination and Validation ............................................................................31 3.3 Overall Structure ................................................................................................................35 3.4 Quaternary interfaces .........................................................................................................35 3.5 Heptad Stutter and Chloride Coordination ........................................................................36 3.6 Structural Comparison of Syncytin-1 and Syncytin-2 TM ................................................39 3.7 Structural Comparison of Syncytin-2 with other CX6CC TMs .........................................45 Chapter 4 Future Directions ...........................................................................................................50 Future Directions .......................................................................................................................50 4.1 Short-term goals .................................................................................................................50 4.2 Conclusions and longer term goals ....................................................................................51 References ......................................................................................................................................53