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I Differential Binding and Structural Properties of the WWC Protein Differential Binding and Structural Properties of the WWC Protein Family by Diego Javier Rodriguez A THESIS Submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degrees of Honors Baccalaureate of Science in Biochemistry and Molecular Biology Honors Baccalaureate of Arts in Psychology (Honors Scholar) Presented June 4, 2020 Commencement June 2020 i AN ABSTRACT OF THE THESIS OF Diego Javier Rodriguez for the degrees of Honors Baccalaureate of Science in Biochemistry and Molecular Biology and Honors Baccalaureate of Arts in Psychology presented on June 4, 2020. Title: Differential Binding and Structural Properties of the WWC Protein Family Abstract approved: ____________________________________________________________ Afua Nyarko The WWC family consists of three proteins‐ KIBRA/WWC1, WWC2, and WWC3. The three proteins share a similar primary structure and interact with a common set of proteins. Each paralog contains two WW domains which recognize proline‐rich sequences of the type PPxY (P is proline, Y is tyrosine, x is any amino acid). WW domains adopt a three‐stranded antiparallel beta sheet fold, but the WWC proteins are “atypical” in that, the second WW domain (WW2) is unfolded. The WWC proteins are implicated in regulating cell proliferation, organ growth, cell migration, synaptic signaling and generally thought to perform overlapping functions in most tissues. However, in recent years, several studies point to unique functions for each paralog. This study presents data that suggest that the unique functions arise from differences in binding mechanisms. Constructs of the WW domains of KIBRA and WWC3 and the PPxY segments of four putative binding partners, Large tumor suppressor 1 (LATS1), Angiomotin-like 1 (AMOTL1), DENDRIN, and Synaptopodin (SYNPO) were cloned and over-expressed in E.coli. Binding affinities and binding-induced structural changes were determined by isothermal titration calorimetry (ITC), fluorescence spectroscopy (FS) and nuclear magnetic resonance spectroscopy (NMR). The ITC data show that the LATS1, DENDRIN, and AMOTL1 polypeptides bind the KIBRA WW domains with a higher affinity than the WWC3 WW domains. The FS and NMR data further show that the binding of LATS1 and SYNPO to KIBRA and WWC3 does not induce ii folding of the WW2 domain, however, mutant LATS1 and SYNPO constructs, designed to mimic the short linker between the DENDRIN PPxY sites, induced folding of the KIBRA and WWC3 WW2 domains. The results suggest that differences in binding affinities for partner proteins and/or binding-induced folding may contribute to the unique functions of the WWC proteins. Key Words: WWC family, WW domains, PPxY motif Corresponding e-mail address: [email protected] iii ©Copyright by Diego Javier Rodriguez June 4, 2020 iv Differential Binding and Structural Properties of the WWC Protein Family by Diego Javier Rodriguez A THESIS Submitted to Oregon State University Honors College in partial fulfillment of the requirements for the degrees of Honors Baccalaureate of Science in Biochemistry and Molecular Biology Honors Baccalaureate of Arts in Psychology (Honors Scholar) Presented June 4, 2020 Commencement June 2020 v Honors Baccalaureate of Science in Biochemistry and Molecular Biology project of Diego Javier Rodriguez presented on June 4, 2020 APPROVED: Afua Nyarko, Mentor, representing Department of Biochemistry and Biophysics Elisar Barbar, Committee Member, representing Department of Biochemistry and Biophysics Ethiene Kwok, Committee Member, representing Department of Biochemistry and Biophysics Patrick Reardon, Committee Member, representing Department of Biochemistry and Biophysics Toni Doolen, Dean, Oregon State University Honors College I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request. Diego Javier Rodriguez, Author vi ACKNOWLEDGEMENT My development as a scientist and my work in this thesis, I owe to Dr. Nyarko and the Nyarko lab, for mentoring me and providing me a space to learn. Every member of the Nyarko Lab took a part of my development. Including the mentorship of Afua and the countless hours we spent working on my projects, posters, discussions, troubleshooting, and this thesis. The patient guidance of Kasie Baker, who was my first introduction into wet lab work, has been immeasurably important to this thesis and my development as a scientist. Not only did she aid me by running, collecting, and processing all the NMR data presented in this thesis, Kasie guided me through protein purification and how to conduct many biophysical experiments. Ethiene ‘Kathy’ Kwok also played an integral part of my development and guided me through the molecular cloning and additional biophysical techniques. And, Amber Vogel was always around for any help I needed, advice, and countless discussions on our projects- past, present, and future. In addition to the Nyarko Lab, the Dept. of Biophysics and Biochemistry provided immeasurable support. Whether, through the tireless work of my advisor Dr. Kari Van Zee or the many ways the department leadership, including Drs. Elisar Barbar, Andy Karplus, and Michael Freitag, helped me succeed. In addition, funding from the Cripp’s Undergraduate Experience Award, the Undergraduate Research and Scholarship Award, and the Honor’s College DeLoach Work Scholarship helped support me and my work presented in this thesis. I would also like to thank my parents for always giving me something when they had nothing. This work and my education would have never happened if it were not for them. Lastly, I want to thank my family and friends who helped support me, made me laugh, and motivated me to always work hard. i Table of Contents List of Figures ................................................................................................................................ iv List of Tables .................................................................................................................................. v List of Abbreviations ..................................................................................................................... vi 1. INTRODUCTION ................................................................................................................... 1 1.1. Functions of the WWC protein family ............................................................................. 1 KIBRA: .................................................................................................................................... 1 WWC2: .................................................................................................................................... 2 WWC3: .................................................................................................................................... 2 1.2. Structure of the WWC protein family .............................................................................. 3 1.3. PPxY-containing binding partners of the WWC proteins ................................................ 5 Dendrin: ................................................................................................................................... 5 Synaptopodin: .......................................................................................................................... 6 Large associated tumor suppressor 1: ...................................................................................... 6 Angiomotin-like 1: .................................................................................................................. 7 2. EXPERIMENTAL DESIGN ................................................................................................... 8 2.1. Cloning of Constructs....................................................................................................... 8 2.2. Recombinant Protein Production ..................................................................................... 9 2.3. Isothermal Titration Calorimetry ................................................................................... 10 2.4. Fluorescence Spectroscopy ............................................................................................ 10 2.5. Circular Dichroism ......................................................................................................... 10 2.6. Nuclear Magnetic Resonance Spectroscopy .................................................................. 11 2.7. X-Ray Crystallography Protein Preparation and Screening ........................................... 11 3. RESULTS .............................................................................................................................. 12 3.1. Design and characterization of constructs ...................................................................... 12 3.2. ITC studies of the WWCs-PPxY partner interactions .................................................... 13 3.3. ITC binding studies of LATS1 and SYNPO mutants .................................................... 14 3.4. Binding-induced folding of the KIBRA WW domains .................................................. 15 3.5. Binding-induced folding of WWC3 WW domains ........................................................ 18 4. DISCUSSION ........................................................................................................................ 19 4.1. KIBRA and
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