Solution Structure and Characterization of Lipid Binding of the Noxo1β Px Domain
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SOLUTION STRUCTURE AND CHARACTERIZATION OF LIPID BINDING OF THE NOXO1β PX DOMAIN By NICOLE YOLANDA DAVIS A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Biochemistry and Molecular Biology Program August 2010 Winston-Salem, North Carolina Approved by: Dr. David A. Horita, Ph.D., Advisor Examining Committee: Dr. Sean Reid, Ph.D., Chair Dr. Tom Hollis, Ph.D. Dr. Greg Kucera, Ph.D. Dr. Linda C. McPhail, Ph.D. ACKNOWLEDGEMENTS I would like to thank my advisor, David Horita and my committee members (Sean Reid, Linda McPhail, Tom Hollis and Greg Kucera) for their help and guidance throughout my time here. I know my project (along with myself) was difficult at times and I thank you for your patience. I would also like to thank some current and former members of the Horita lab. Joel, I cannot thank you enough for the work you did early on with NOXO1. You helped push the project along while I worked on other projects. I appreciate all the times you immediately stopped to answer any questions I had in lab. Kai, I want to thank you for the little bits of encouragement you gave me. When times were really stressful and frustrating, remembering times when you congratulated me for what seemed to be the most mundane task helped me push through. Lindsay, I am sad we did not get more time together in lab, but I am glad we got to know each other. To all of my friends: I would never have made it through graduate school without you. From personal to professional, I cannot thank you enough for your constant support. To H!, you know too much, we have to stay friends forever. To the martini night girls (Keri, Carla, Heather and Karon), I will miss the conversations and $5 martinis we had over the summer, they are times I will always fondly remember. To E, you are a sick man, SICK! but I thank you for being there for me in times of laughter and times of tears. To Joy, thank you for all of the useful and humorous career advice you shared with me. I still believe you need to write a book on life post-grad school. Erin, thank you for simultaneously telling people I am your smartest friend yet treating me like I’m a normal person. You are always someone I look to for advice and you have never let me down. To Valerie and Amanda, thanks for the constant emails, gossip and vacation plans. I am glad we’ve reconnected since high school. I would like to thank Jim Gaffigan for being a constant source of inspiration, humor…and bacon. To my parents, thank you for paying for the first fourteen years of school while I “paid” for the last eleven. Thank you for letting me be who I am, for supporting me and being my biggest cheerleaders. I hope I have made you proud. To my mom thank you for trying to understand what I am researching; you understand it more than you realize. To my dad, I want to thank you for asking me random questions dealing with any area of science and thinking I should know the answer. To my brother, I’ve enjoyed getting to know you as an adult and learning we share a similar, twisted sense of humor. I also thank you for succinctly stating that all I do is just stick goo in a magnet. ii TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS…………….……………………………………………...iv LIST OF ABBREVIATIONS…………………...............................................................vi ABSTRACT……………………………………………………………………………...x CHAPTER I. INTRODUCTION……………………………………………….1 CHAPTER II. NOXO1β PX BINDS TO PI(4,5)P2 IN ADDITION TO NEUTRAL MEMBRANE LIPIDS……………………………..29 CHAPTER III. SOLUTION STRUCTURE OF NOXO1β PX………….………76 CHAPTER IV. DISCUSSION…………………………………………………..131 APPENDIX……………………………………………………………………………..142 CURRICULUM VITAE………………………………………………………………..152 iii LIST OF ILLUSTRATIONS CHAPTER I Page Figure 1. Components and Domain Structure of the Phagocytic NADPH 5 Oxidase Figure 2. NADPH Oxidase Isoforms 10 Figure 3. Domain Comparison of NOXO1 and p47phox and Alignment of 15 NOXO1 PX Isoforms CHAPTER II Figure 1. Dot Blots for NOXO1β PX, p40phox PX and p47phox PX 43 Figure 2. SDS-PAGE Gel of GST-NOXO1β PX Binding to PA LUVs. 46 Figure 3. 12% SDS-PAGE Gel of NOXO1β PX Binding to PI(5)P LUVs 49 Figure 4. The Magnetic Bead Assay is not a Reliable Method for 51 Measuring Lipid Binding Figure 5. PA LUVs Do Not Dissociate from a GST-p47phox PX Coated 54 Surface Figure 6. Increasing the NaCl Concentration Decreased Binding of 58 NOXO1β PX to a POPC/POPE Surface Figure 7. The Addition of Phosphate Decreases Binding of NOXO1β 60 PX to a POPE/POPC Surface Figure 8. The Addition of Phosphate Decreases Binding of NOXO1β 62 PX to a 3% PI(4,5)P2 Surface Figure 9. NOXO1β PX Binds to Background Phospholipids 65 Figure 10. NOXO1β PX Binds to PI(4,5)P2 67 phox phox Figure 11. P40 PX and p47 PX bind to PI(3)P and PI(3,4)P2 Respectively 70 Figure 12. NOXO1β PX Does Not Bind to Low Levels of Anionic Lipids 72 iv CHAPTER III Figure 1. 15N, 1H HSQC of NOXO1β PX 88 Figure 2. Predicted secondary structure of NOXO1β PX by TALOS+ 90 Figure 3. NOXO1β PX is a monomer in solution 93 Figure 4. Solution Structure of NOXO1β PX 96 Table 1. NOXO1β PX Restraints and Structure Statistics 98 2 Figure 5. S , τe and Rex of NOXO1β PX 102 Figure 6. NOXO1β PX does not have a stable core 105 Figure 7. POPC Nanodiscs are a suitable membrane mimetic for 108 NOXO1β PX Figure 8. NOXO1β PX exhibits non-specific binding to a POPC nanodisc 110 Figure 9. NOXO1β PX diC8-PI(4,5)P2 titration 113 Figure 10. NOXO1β PX does not undergo many chemical shift changes 116 upon addition of diC4-PI(3,4)P2 Figure 11. NOXO1β PX undergoes chemical shift changes upon addition 119 of diC4-PI(4,5)P2 Figure 12. NOXO1β PX does not contain conserved residues for binding to PIs 122 phosphorylated at the D3 position Figure 13. NOXO1β PX contains conserved residues for binding to PIs 125 phosphorylated at the D4 position Figure 14. Candidate residues of NOXO1β PX for binding to PIs 128 phosphorylated at the D5 position APPENDIX Figure 1. Domain Organization of RIP1 and Constructs of RIP1 148 Used For Binding Assays v LIST OF ABBREVIATIONS AIR, autoinhibitory region ApoA1, Apolipoprotein A-1 BSA, bovine serum albumin CGD, chronic granulomatous disease CHAPS, (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate CISK, cytokine-independent survival kinase CMC, critical micelle concentration DCN, 2H13C15N isotopically labeled protein DCN-ILV, 2H13C15N-1H(Iδ1,L,V) isotopically labeled protein DCN-ILV-F, 2H13C15N-1H(Iδ1,L,V)-15N Phe isotopically labeled protein DD, death domain DHPC, 1,2-dihexanoyl-sn-glycero-3-phosphocholine DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine DN, 2H15N isotopically labeled protein DUOX(1-2), dual oxidase (1-2) E. coli, Escherichia coli FAD, flavin adenine dinucleotide gp91phox (NOX2), 91-kDa phagocytic oxidase component GST, glutathione S transferase ID, intermediate domain IP3, inositol-3-phosphate vi KD, kinase domain LPA, lysophosphatidic acid LPC, lysophosphatidyl choline LUV, large unilamellar vesicles MAPK, mitogen activated protein kinase NADPH, nicotinamide adenine dinucleotide phosphate NMR, Nuclear Magnetic Resonance NOX(1-5), NADPH oxidase (1-5) NOXA1, NADPH oxidase activator 1 NOXO1, NADPH oxidase organizer 1 - O2 , superoxide anion OG, n-octyl-β-D-glucopyranoside p22phox, 22-kDa phagocytic oxidase component p40phox, 40-kDa phagocytic oxidase component p47phox, 47-kDa phagocytic oxidase component p67phox, 67-kDa phagocytic oxidase component PA, phosphatidic acid PB1, PHOX Bem1 domain PBS, phosphate buffered saline PC, phosphatidylcholine PE, phosphatidylethanolamine PG, phosphatidylglycerol PHOX, phagocytic oxidase vii PI, phosphatidylinositols PI(3)P, phosphatidylinositol-3-phosphate PI(4)P, phosphatidylinositol-4-phosphate PI(5)P, phosphatidylinositol-5-phosphate PI(3,4)P2, phosphatidylinositol-3,4-bisphosphate PI(3,5)P2, phosphatidylinositol-3,5-bisphosphate PI(4,5)P2, phosphatidylinositol-4,5-bisphosphate PI(3,4,5)P3, phosphatidylinositol-3,4,5-triphosphate PI3K, phosphatidylinositol-3-kinase PIP, phosphatidylinositol phosphate PLD, phospholipase D PR, proline-rich-region PS, phosphatidylserine PX, phox homology RHIM, receptor interacting protein homotypic interaction motif RIP1, receptor interacting protein 1 ROS, reactive oxygen species RU, response units S1P, sphingosine-1-phosphate SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis SH3, Src homology 3 siRNA, small interfering RNA S/N, signal-to-noise viii SPR, surface plasmon resonance TBST, Tris-buffered saline with Tween Tks4, tyrosine kinase substrate with four SH3 domains Tks5, tyrosine kinase substrate with five SH3 domains TNF, tumor necrosis factor TNFR1, tumor necrosis factor receptor 1 TRADD, tumor necrosis factor receptor 1 associated death domain protein TRAF2, tumor necrosis factor receptor 1 associated factor 2 TROSY, transverse relaxation-optimized spectroscopy Trx, thioredoxin ix ABSTRACT Nicole Y. Davis SOLUTION STRUCTURE AND CHARACTERIZATION OF LIPID BINDING OF THE NOXO1β PX DOMAIN Dissertation under the direction of David A. Horita, Ph.D., Associate Professor of Biochemistry The NADPH oxidases are multiprotein enzyme complexes which catalyze the formation of reactive oxygen species (ROS). The most studied of these is the phagocytic NADPH oxidase (PHOX), which is found in neutrophils, macrophages and monocytes. The PHOX enzyme plays a role in the host immune response through production of ROS. Deficiencies in components of the phagocytic NADPH oxidase lead to weakened host defense, as seen in chronic granulomatous disease. There are also non-phagocytic homologues of the phagocytic NADPH oxidase (NOX) found in a broad range of tissues.