Brigham Young University BYU ScholarsArchive All Theses and Dissertations 2016-12-01 Structural Analysis of Cell Signaling Complexes Takuma Aoba Brigham Young University Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Chemistry Commons BYU ScholarsArchive Citation Aoba, Takuma, "Structural Analysis of Cell Signaling Complexes" (2016). All Theses and Dissertations. 6583. https://scholarsarchive.byu.edu/etd/6583 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in All Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Structural Analysis of Cell Signaling Complexes Takuma Aoba A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Barry M. Willardson, Chair Gregory F. Burton Joshua L. Andersen Kenneth A. Christensen Joshua L. Price Department of Chemistry and Biochemistry Brigham Young University Copyright © 2016 Takuma Aoba All Rights Reserved ABSTRACT Structural Analysis of Cell Signaling Complexes Takuma Aoba Department of Chemistry and Biochemistry, BYU Doctor of Philosophy of Science Bardet-Biedl syndrome (BBS) is a rare genetic disease that causes retinal degradation, obesity, kidney dysfunction, polydactyly, and other cilium-related disorders. To date, more than 20 BBS genes, whose mutants cause BBS phenotypes, have been identified, and eight of those (BBS1-2, 4-5, 7-9, and 18) are known to form the BBSome complex. Recent studies have revealed that the BBSome is closely involved in the trafficking of signaling proteins in the primary cilium. Mutations in BBS genes are highly pathogenic because trafficking in the primary cilium is not fully functional when BBS mutations impair assembly of the BBSome. However, the functional links between onset of BBS and BBSome assembly are not well understood. To address this gap in knowledge, we examined the structure of a BBSome assembly intermediate, the BBSome core complex (BBS2, 7, and 9). We employed a combination of chemical crosslinking coupled with mass spectrometry (XL-MS) and electron microscopy (EM) to determine the structure. We applied this structural information to BBS mutations in the core complex to understand how these mutations might cause the disease. These results provide the first structural model of the BBSome core complex and give insight into the molecular basis of Bardet-Biedl syndrome. We have also investigated the mechanism of assembly of the two mTOR kinase complexes (mTORC1 and 2). mTOR is a master regulator of cell metabolism, growth and proliferation. As such, mTOR is a high-value drug target. We investigated the mechanism of assembly of these mTOR complexes and found that the cytosolic chaperonin CCT contributes to mTOR signaling by assisting in the folding of mLST8 and Raptor, components of mTORC1 and mTORC2. To understand the function of CCT in mTOR complex assembly at the molecular level, we have isolated the mLST8-CCT complex and performed a structural analysis using chemical cross- linking couple with mass spectrometry (XL-MS) and cryogenic EM. We found that mLST8 binds CCT deep in its folding cavity, making specific contacts with the CCTα and γ subunits and forming a near-native β-propeller conformation. This information can be used to develop new therapeutics that regulate mTOR activity by controlling mTOR complex assembly. Keywords: BBS, primary cilia, BBSome core complex, EM, XL-MS, CCT, mTORC, PhLP1 ACKNOWLEDGEMENTS I extend much appreciation to Dr. Willardson, who was insightful and a great mentor throughout my graduate studentship. Besides his responsibility as a professor of biochemistry, advisor of many other students, and principal investigator, he sacrificed a significant amount of time helping me compose and refine the present dissertation. Other members of his lab, Grant Ludlum and Madhura Dhavale made significant contribution to the findings presented herein as well. It is certain that I would not have been able to accomplish this work without collaboration of José Valpuesta’s lab at the Centro Nacional de Biotecnología in Madrid and Sarah Franklin’s lab at the University of Utah. I would like to give my deepest gratitude to my daughters and my wife, Akiko, who were always faithful, patient, and supportive in unaccustomed cultures during my graduate research. TABLE OF CONTENTS TITLE PAGE ................................................................................................................................... i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF FIGURES ...................................................................................................................... vii ABBREVIATIONS ..................................................................................................................... viii CHAPTER 1: BBS AND BBSOME................................................................................................1 Summary ......................................................................................................................................1 Introduction ..................................................................................................................................2 Bardet-Biedl syndrome .............................................................................................................2 BBS genes ................................................................................................................................3 Cilium as cellular antenna ........................................................................................................4 Physiological role of the BBSome ...........................................................................................6 Assembly of the BBSome ........................................................................................................9 Homology between BBSome core subunits and COP proteins ..............................................11 Cryogenic electron microscopy ..............................................................................................12 Chemical crosslinking coupled with mass spectrometry ........................................................12 Unnatural amino acid photo-crosslinking ..............................................................................15 Conclusion ..................................................................................................................................17 CHAPTER 2: STRUCTURAL ANALYSIS OF THE BBSOME CORE COMPLEX .................19 Summary ....................................................................................................................................19 Introduction ................................................................................................................................20 Methods ......................................................................................................................................22 Purification of the BBSCC .....................................................................................................22 Crosslinking coupled with mass spectrometry .......................................................................23 Mass spectrometry ..................................................................................................................24 XL-MS analysis ......................................................................................................................25 Unnatural amino acid crosslinking .........................................................................................25 Mutagenesis ............................................................................................................................27 Protein separation for EM sampling .......................................................................................28 iv EM grid preparation and data collection ................................................................................28 Image processing, particle selection and 2D classification ....................................................29 3D reconstruction ...................................................................................................................29 Generating a structural model ................................................................................................29 Results ........................................................................................................................................30 Purification of the BBSCC .....................................................................................................30 Electron microscopy ...............................................................................................................31 Structures of BBS2, 7, and 9 domains ....................................................................................32 BBS2 and BBS7 interact through a coiled-coil domain .........................................................34