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A Study of Conserved Peptide-Binding Domains Western Washington University Western CEDAR WWU Graduate School Collection WWU Graduate and Undergraduate Scholarship Spring 2021 Signaling and Trafficking in Choanoflagellates and Humans: A Study of Conserved Peptide-Binding Domains Haley Wofford Western Washington University, [email protected] Follow this and additional works at: https://cedar.wwu.edu/wwuet Part of the Chemistry Commons Recommended Citation Wofford, Haley, "Signaling and Trafficking in Choanoflagellates and Humans: A Study of Conserved Peptide-Binding Domains" (2021). WWU Graduate School Collection. 1032. https://cedar.wwu.edu/wwuet/1032 This Masters Thesis is brought to you for free and open access by the WWU Graduate and Undergraduate Scholarship at Western CEDAR. It has been accepted for inclusion in WWU Graduate School Collection by an authorized administrator of Western CEDAR. For more information, please contact [email protected]. Signaling and Trafficking in Choanoflagellates and Humans: A Study of Conserved Peptide-Binding Domains By Haley Wofford Accepted in Partial Completion of the Requirements for the Degree Master of Science ADVISORY COMMITTEE Dr. Jeanine Amacher, Chair Dr. P. Clint Spiegel Dr. Sergey Smirnov GRADUATE SCHOOL Dr. David L. Patrick, Dean Master’s Thesis In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Western Washington University, I grant to Western Washington University the non-exclusive royalty-free right to archive, reproduce, distribute, and display the thesis in any and all forms, including electronic format, via any digital library mechanisms maintained by WWU. I represent and warrant this is my original work, and does not infringe or violate any rights of others. I warrant that I have obtained written permissions from the owner of any third party copyrighted material included in these files. I acknowledge that I retain ownership rights to the copyright of this work, including but not limited to the right to use all or part of this work in future works, such as articles or books. Library users are granted permission for individual, research and non-commercial reproduction of this work for educational purposes only. Any further digital posting of this document requires specific permission from the author. Any copying or publication of this thesis for commercial purposes, or for financial gain, is not allowed without my written permission. Haley Wofford May 28, 2021 Signaling and Trafficking in Choanoflagellates and Humans: A Study of Conserved Peptide-Binding Domains A Thesis Presented to The Faculty of Western Washington University In Partial Fulfillment Of the Requirements for the Degree Master of Science by Haley Wofford May 2021 Abstract Many crucial cellular processes are regulated by signaling and trafficking pathways, which are largely dependent on protein interactions in the cell. These networks are thought to play a crucial role in the evolution of multicellular organisms from their unicellular ancestors. Choanoflagellates are unicellular organisms that can adopt a multicellular state, called a rosette. Though they evolved independently from and prior to the diversification of metazoans, they are the closest extant nonmetazoan ancestor to animals, making them a compelling model for the study of early multicellularity. Here, we look at two structurally conserved peptide-binding domains that both play important roles in cellular signaling and trafficking. PDZ and SH2 domains are peptide-binding domains that have highly conserved structures and are found in humans and choanoflagellates. We analyzed the binding cleft of a non-homologous PDZ domain and compared the binding affinities of that PDZ domain to two human PDZ domains. We also developed a program that looks specifically at the PDZ binding cleft and finds the most similar human PDZ domain to an uncharacterized choanoflagellate PDZ domain, then finds potential endogenous target sequences of the uncharacterized PDZ domain. To assess conservation of PDZ domain structure we solved high-resolution crystal structures of a representative M. brevicollis PDZ domain that is homologous to human Dlg1 PDZ2. We also studied the binding cleft of a conserved SH2 domain from the TEC family of kinases. Overall, we find that interactions between the domain and non-motif residues dramatically influence peptide binding and, using our program, we can begin to predict successful interactions. The evolutionary conservation of PDZ and SH2 domain makes them ideal candidates to study to better understand how the evolution of signaling and trafficking domains and pathways coincided with the emergence of multicellularity. iv Acknowledgements Over the course of the research for and writing of this thesis I have received a great deal of support, assistance, and encouragement. First, I would like to thank my advisor, Dr. Jeanine Amacher, whose expertise and insight was crucial in formulating the research questions and study designs herein. Your feedback allowed me to become not only a better scientist, but a better critical thinker. I would like to acknowledge the other members of my thesis committee, Dr. Clint Spiegel and Dr. Sergey Smirnov, whose guidance was invaluable in the completion of this thesis. Thank you both. Thank you to all members of the Amacher Lab who contributed to this project. Special thanks to Kevin Alexander Estrada Alamo who helped purify proteins, as well as Brandon Vogel and Frederick Longshore-Neate who contributed to the computational aspects of this project. Josh Myers-Dean and Filip Jagodzinski wrote the program described herein and were instrumental in bringing that idea to life. It has been an absolute pleasure working with you both. Thank you for a wonderful collaboration. I would like to thank my biochemistry cohort: AP Wang, Erin Rosenkranz, Micah Nakao, and Shaun Peters; as well was Dr. Kenneth Childers and Chris Swanson. You all provided stimulating discussions, wise insight, and the best listening ears anyone could ask for. Thank you all for your friendship. Thank you to the Chemistry Department at Western Washington University for all your help: Dr. James Vyvyan, Sam Danforth, Amanda Weiss, Gary Carlton, Steve Sible, Miranda Abrashi, and Alexi Guddal. I would also like to thank the NSF and Western Washington University who funded my research and teaching assistantships for my graduate studies. In addition, I would like to thank my parents, who taught me to work hard and stay curious. Finally, my sister, who has always been, and will always be, my biggest inspiration. v Table of Contents Abstract ......................................................................................................................................... iv Acknowledgements ....................................................................................................................... v List of Tables and Figures .......................................................................................................... vii Introduction ................................................................................................................................... 1 Chapter 1: Biochemical characterization and computational analysis of non-homologous PDZ domains in Monosiga brevicollis ......................................................................................... 4 1.1 Introduction: PDZ Domains ................................................................................................ 5 1.2 Results................................................................................................................................... 8 1.2.1 Structural and biochemical characterization of A9UPE9 PDZ. ................................... 8 1.2.2 Binding experiments of A9UPE9 PDZ and DLG1 PDZ1. ............................................. 9 1.2.3 Structural and biochemical characterization of A9V7X5 PDZ. .................................. 17 1.3 Concluding Remarks.......................................................................................................... 20 1.4 Materials and Methods ...................................................................................................... 22 1.4.1 Protein Expression and Purification. .......................................................................... 22 1.4.2 Crystallization and Data Collection. ........................................................................... 23 1.4.3 Binding assays by fluorescence polarization. .............................................................. 24 Chapter 2: Structural characterization of DLG homolog Monosiga brevicollis A9UT73 PDZ2............................................................................................................................................. 25 2.1 Results: Structural characterization of PDZ domains in mbDLG ................................... 26 2.2 Concluding Remarks.......................................................................................................... 30 2.3 Materials and Methods ...................................................................................................... 33 2.3.1 Protein expression and purification ............................................................................. 33 2.3.2 Crystallization, data collection, and structure determination ..................................... 34 Chapter 3: A study of homologous TEC family kinase SH2 domains ................................... 35 3.1 Introduction: TEC family kinases and
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