STRUCTURE AND REGULATION OF YEAST GLYCOGEN SYNTHASE Sulochanadevi Baskaran Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Biochemistry and Molecular Biology Indiana University August 2010 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Thomas D. Hurley, Ph.D., Chair Anna A. DePaoli-Roach, Ph.D. Doctoral Committee Millie M. Georgiadis, Ph.D. Peter J. Roach, Ph.D. May 19, 2010 William J. Sullivan Jr., Ph.D. ii Dedication I dedicate this work to my family, friends and mentors, for their support, encouragement, inspiration and guidance. iii Acknowledgements The work presented here is a result of the collaborative effort of many people and it is my pleasure to thank everyone who made this possible. First and foremost, I am indebted to my advisor, Dr. Thomas Hurley, for his invaluable scientific guidance, enthusiastic supervision and encouragement throughout my thesis research work. I am very grateful for his support, patience and his efforts in explaining and teaching me the concepts of macromolecular crystallography. I will always be thankful to him for teaching me the most important factor that is required for scientific research - perseverance. I would like to express my sincere gratitude to my committee members Drs. Peter Roach, Anna DePaoli-Roach, Millie Georgiadis and William Sullivan for their time, support, guidance and helpful suggestions. I am thankful to lab colleagues Dr. Samantha Perez-Miller, Dr. Heather Larson, Dr. Jianzhong Zhou, Dr. Lillian González-Segura, Ram Vanam, Jason Braid, Lili Zang, Bibek Parajuli, Dr. May Khanna and Dr. Ann Kimble-Hill for providing a stimulating and fun environment in the lab. I am especially grateful to Dr. Samantha Perez-Miller and Dr. Heather Larson for their friendship and support. I appreciate the help of my colleagues, Dr. Kristie Goodwin, Sarah Delaplane, Dr. Hongzhen He, Dr. Tsuyoshi Imasaki, Dr. Alexander Skurat, Dr. Jose Irimia-Dominguez, Vinnie Tagliabracci, Sixin Jiang, Cathy Meyer, Dyann Segvich and Chandra Karthik. I would like to extend my thanks to Drs. Yuichiro Takagi, Mark Goebl, Ronald Wek and Zhong-Yin Zhang for their encouragement and to the biochemistry office staff for their timely help and assistance. iv I wish to thank the beamline scientist at the SBC-CAT and GM/CA-CAT stations at the Advanced Photon Source, in particular Drs. Stephan Ginell, Norma Duke, Marianne Cuff, Nagarajan Venugopalan and Michael Becker for their scientific assistance. I would like to thank Dr. Lou Messerle for providing the tantalum cluster compound and Dr. Thomas Terwilliger for sending the scripts for partial model phasing. I am grateful to my teachers - Drs. Bhaskar-Rao, Sairam, Illango and Dharmalingam for encouraging me to pursue a career in scientific research. I would like to specifically thank Drs. Usha, Krishnaswamy and Mohammed Rafi for introducing me to the world of structural biology and macromolecular crystallography. I would like to thank my friends Dr. Shankar Varadarajan, Sowmya Chandrasekar, Dr. Raji Muthukrishanan, Sirisha Pochareddy, Dr. Judy Rose James and Dr. Aditi Bapat for their emotional support and helping me get through the difficult times. Finally I would like to express my sincere thanks to my family for everything they had provided me all through my life. v Abstract Sulochanadevi Baskaran STRUCTURE AND REGULATION OF YEAST GLYCOGEN SYNTHASE Glycogen is a major energy reserve in most eukaryotes and its rate of synthesis is controlled by glycogen synthase. The activity of eukaryotic glycogen synthase is regulated by the allosteric activator glucose-6-phosphate, which can overcome the inhibitory effects of phosphorylation. The effects of phosphorylation and glucose-6-phosphate on glycogen synthase are mediated by a cluster of six arginines located within a stretch of 12 amino acids near the C-terminus of the enzyme’s polypeptide chain. We studied isoform-2 of yeast glycogen synthase as a model to study the structural and molecular mechanisms that underlie the regulation of the eukaryotic enzymes and our primary tools of investigation were macromolecular X-ray crystallography, site-directed mutagenesis, intein- mediated peptide ligation and enzyme assays. We have solved the tetrameric structure of the yeast enzyme in two different activity states; the resting enzyme and the activated state when complexed with glucose-6-phosphate. Binding of glucose-6-phosphate to glycogen synthase induces large conformational changes that free the active site of the subunits to undergo conformational changes necessary to catalyze the reaction. Further, using site directed mutagenesis and intein-mediated peptide ligation to create specific phosphorylation states of the enzyme we were able to define specific roles for vi the arginine residues that mediate the regulatory effects of phosphorylation and glucose-6-phosphate activation. Based on these studies, we propose a three state structural model for the regulation of the enzyme, which relate the observed conformational states to specific activity levels. In addition to these regulatory studies, we have also solved the structure of the enzyme complexed with UDP and with substrate analogs, which provide detailed insight into the catalytic mechanism of the enzyme and the ability of glycogen synthase to remain tightly bound to its substrate glycogen. Thomas D. Hurley, Ph.D., Chair . vii TABLE OF CONTENTS LIST OF TABLES ................................................................................................ xii LIST OF FIGURES ............................................................................................. xiii LIST OF ABBREVIATIONS ................................................................................ xvi INTRODUCTION .................................................................................................. 1 A. Glycogen ...................................................................................................... 1 1. Structure of glycogen ................................................................................. 1 2. Biosynthesis and degradation ................................................................... 2 3. Physiological role of glycogen in mammals ............................................... 5 4. Physiological role of glycogen in yeast ...................................................... 9 B. Glycogen synthase ..................................................................................... 11 1. Glycogen storage disease type-0 ............................................................ 11 2. Enzymatic activity of GS .......................................................................... 12 3. Catalytic mechanism of GS ..................................................................... 13 4. Influence of substrate on GS activity ....................................................... 16 5. Regulation of GS activity ......................................................................... 17 6. Structural classification of GS .................................................................. 24 C. Rationale and overview of the thesis research ........................................... 26 D. Theory of experimental methods used ....................................................... 27 1. Macromolecular x-ray crystallography ..................................................... 27 2. Intein mediated peptide ligation ............................................................... 38 METHODS.......................................................................................................... 40 A. Gsy2p wild-type and mutant expression constructs .................................... 40 1. Site-directed mutagenesis ....................................................................... 40 viii 2. Cloning of Gsy2p in the IMPACT vector .................................................. 41 B. Peptide synthesis ....................................................................................... 41 C. Expression and purification of Yeast Gsy2p ............................................... 42 1. Protein preparation from pET28A constructs ........................................... 42 2. Purification of Gsy2p core and semi-synthetic enzymes ......................... 43 D. Crystallization of Gsy2p .............................................................................. 45 1. R580A/R581A/R583A crystals................................................................. 45 2. R589A/R592A glucose-6-phosphate Co-crystals .................................... 46 3. Heavy atom and ligand soaks ................................................................. 47 E. Data collection, processing, structure solution and refinement ................... 48 1. Data collection ......................................................................................... 48 2. Structure solution, model building and refinement ................................... 49 F. Structure analysis ....................................................................................... 50 1. Protein surface analysis .......................................................................... 50 2. Domain rotation analysis ......................................................................... 51 G. GS activity measurement and data analysis .............................................. 51 1. Preparation