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Understanding Binding-Induced Disorder-To-Order and Conformational UNDERSTANDING BINDING-INDUCED DISORDER-TO-ORDER AND CONFORMATIONAL TRANSITIONS IN PROTEINS THAT REGULATE AUTOPHAGY A Dissertation Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Karen Marie Glover In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Department: Chemistry and Biochemistry June 2018 Fargo, North Dakota North Dakota State University Graduate School Title UNDERSTANDING BINDING-INDUCED DISORDER-TO-ORDER AND CONFORMATIONAL TRANSITIONS IN PROTEINS THAT REGULATE AUTOPHAGY By Karen Marie Glover The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of DOCTOR OF PHILOSOPHY SUPERVISORY COMMITTEE: Dr. Sangita Sinha Chair Dr. Gregory Cook Dr. Stuart Haring Dr. Penelope Gibbs Approved: 26 June 2018 Dr. Gregory Cook Date Department Chair ABSTRACT Autophagy, a cellular homeostasis process that degrades and recycles cytosolic contents, is regulated at various stages by protein interactions. BECN1 regulates autophagy through interactions with diverse proteins via binding-induced conformational changes in its intrinsically disordered region (IDR) and flexible domains. Understanding the structure-function relationship of conformational transitions provides a basis for understanding these interactions and may be useful for targeting therapeutics for these proteins. We devised a method to identify IDRs likely to become helical upon binding, even in the absence of known binding partners, and analyzed BECN1 interactions to further understand how binding- induced conformational changes affect BECN1-mediated autophagy regulation. We showed that the BECN1 coiled-coil domain (CCD) and β-α repeated, autophagy-specific domain (BARAD) share an overlapping helix (OH) region that transiently adopts mutually exclusive packing states and transiently packs against the CCD and BARAD, but predominantly packs against the BARAD in the homodimer, and demonstrated that mutation of OH residues that pack against both domains abrogates starvation-induced up-regulation of autophagy. Our results have important implications for the relative stability of autophagy-inactive and autophagy-active BECN1 complexes. We also attempted to establish the mechanism by which various virus-encoded proteins regulate autophagy. Nef, a critical protein for HIV pathogenesis, interacts with BECN1 BARAD regions that pack against the OH. However, we show that Nef does not appear to interact directly with the BECN1 homodimer, suggesting that packing of the OH against the BARAD may inhibit the Nef interaction. Several anti-apoptotic viral BCL2 (vBCL2) proteins down-regulate autophagy by binding the BECN1 BH3 homology domain (BH3D) and mediating binding-induced helical transitions in the BH3D. Although protein purification issues prevented analysis of Kaposi’s sarcoma herpesvirus (KSHV) vBCL2 interactions, we determined that BHRF1, an Epstein-Barr virus (EBV)-encoded vBCL2, down-regulates autophagy by binding a flexible region of BECN1 that includes the BH3D. Further, we solved the 2.6 Å crystal structure of BHRF1 bound to the BID BH3D, a pro-apoptotic BCL2, and showed that the BID BH3D binds ~400-times tighter than the BECN1 BH3D to BHRF1 and inhibits BHRF1-mediated down- regulation of autophagy. iii Together, our studies highlight the importance of binding-induced conformational changes in proteins that regulate autophagy. iv ACKNOWLEDGEMENTS To my advisor, Dr. Sangita Sinha, I express my gratitude for her generous guidance, support, and mentorship. I also thank my committee members: Dr. Gregory Cook, Dr. Stuart Haring, and Dr. Penelope Gibbs for their advice and direction. I also extend thanks to our collaborator Dr. Christopher Colbert at NDSU for generously sharing his expertise, advice, and support, and our other collaborators, especially: Dr. Srinivas Chakravarthy at Bio-CAT, APS, Argonne. I am further grateful to current and former members of the Sinha group, including Yang Mei, Minfei Su, Yue Li, Shreya Mukhopadhyay, Srinivas Dasanna, Elizabeth Bueno, and Samuel Wyatt, and the Colbert group, including Jaime Jensen, Shane Wyborny, Benjamin LeVahn, and Beau Jernberg. Thank you for constant advice, assistance, and encouragement. This research was supported an funded by NIH grants RO3 NS090939, R15 GM122035 (S.S.), P20 RR015566 (S.S), R21 AI078198 (S.S), and R15 GM113227 (C.C.); a NSF grant MCB-1413525 (S.S.); a ND Dept. of Commerce Award #14-11-J1-73 (S.S), a NDSU Graduate School doctoral dissertation award (K.G.), and a ND EPSCoR doctoral dissertation award (K.G., S.S.). Work performed at Bio-CAT was supported by NIH NIGMS 9P41 GM103622 and use of the Pilatus 3 1M detector funded by NIH NIGMS 1S10OD018090-01. Work performed at NE-CAT was supported by NIH NIGMS P41 GM103403. This research used resources of the APS, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02- 06CH11357. This research used resources of the Advanced Photon Source, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. I also acknowledge the NDSU Core Biology Facility (funded by NIH grant P30 GM103332-01) for access to tissue culture facilities and the NDSU Advanced Imaging and Microscopy Core Laboratory for access to microscopy and imaging equipment. I also thank Dr. Pawel Borowicz for assistance with imaging and puncta quantification method development. v DEDICATION For my husband Josh, who is my favorite. For my daughter Indiana, who inspires me to be the best version of myself. For my parents, who encourage me in all things great and small. And for my daughter, who wrote this with me. vi TABLE OF CONTENTS ABSTRACT .................................................................................................................................................. iii ACKNOWLEDGEMENTS ............................................................................................................................ v DEDICATION ............................................................................................................................................... vi LIST OF TABLES ........................................................................................................................................ xii LIST OF FIGURES .................................................................................................................................... xiii LIST OF ABBREVIATIONS ...................................................................................................................... xvii CHAPTER 1. INTRODUCTION ................................................................................................................... 1 1.1. BECN1 in autophagy .......................................................................................................... 1 1.1.1. BCL2 binding to BECN1 ........................................................................................ 3 1.1.2. BECN1 in PI3KC3 complexes ............................................................................... 4 1.2. Viral evasion and subversion of autophagy ....................................................................... 5 1.3. Overview of methods used in this research ....................................................................... 6 1.3.1. Cellular autophagy assays .................................................................................... 6 1.3.2. Isothermal titration calorimetry (ITC) ..................................................................... 7 1.3.3. Circular dichroism (CD) spectroscopy ................................................................... 9 1.3.4. Small angle X-ray scattering (SAXS) .................................................................. 10 1.3.5. Protein X-ray crystallography .............................................................................. 13 1.4. Specific aims of this research .......................................................................................... 16 CHAPTER 2. IDENTIFYING INTRINSICALLY DISORDERED PROTEIN REGIONS LIKELY TO UNDERGO BINDING-INDUCED HELICAL TRANSITIONS ...................................................................... 17 2.1. Introduction ...................................................................................................................... 17 2.2. Materials and methods ..................................................................................................... 18 2.2.1. Production of IDRs .............................................................................................. 18 2.2.2. Prediction of disorder and likelihood of folding upon binding .............................. 19 2.2.3. CD spectroscopy ................................................................................................. 19 2.3. Results ............................................................................................................................. 20 2.3.1. Selection of IDRs for analysis ............................................................................. 20 vii 2.3.2. Prediction of IDRs likely to fold upon binding ...................................................... 22 2.3.3. All the selected IDRs lack α-helical structure in solution ....................................
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