Biophysical Studies of the First Nucleotide Binding Domain of Sulfonylurea Receptor 2A to Assess the Significance of Phosphorylation and Mutations by Elvin Dominic de Araujo A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Chemistry University of Toronto ©Copyright by Elvin Dominic de Araujo 2015 Biophysical Studies of the First Nucleotide Binding Domain of Sulfonylurea Receptor 2A to Assess the Significance of Phosphorylation and Mutations Elvin Dominic de Araujo Doctor of Philosophy Department of Chemistry University of Toronto 2015 Abstract The sulfonylurea receptor 2A (SUR2A) proteins are ATP-binding cassette (ABC) transporters that form regulatory subunits in ATP-sensitive potassium channels (K ATP ) channels found in metabolically active tissues. In K ATP channels, four SUR proteins surround four pore-forming Kir6 subunits. By sensing intracellular [ATP]/[ADP] ratios, KATP channels couple the metabolic state of the cell to the membrane potential and therefore have crucial roles in many biological processes. For example, K ATP channels in the pancreas are vital for proper insulin regulation, whereas cardiac K ATP channels contribute to shortening of action potentials which may protect the heart against arrhythmias. Gating of K ATP channels is a complex process that involves multiple ligands and protein domains. ATP binding at the Kir subunits closes the pore, whereas MgATP binding and hydrolysis at the SUR nucleotide binding domains (NBDs) results in channel opening. Thus, the NBDs are critical sites of regulation for the K ATP channel, although the molecular mechanisms for how the NBDs alter channel activity is not well understood. This is mainly a consequence of poor solubility of the isolated SUR NBDs. Here we have developed various strategies that have allowed us to perform detailed NMR experiments and have provided molecular level information ii on the conformation of SUR2A NBD1. These studies enabled us to investigate the structural, dynamic and functional effects of phosphorylation, disease-causing mutations and drug binding in NBD1. Our data suggest that SUR2A NBD1 contains a number of disordered loops that may function as regulatory regions. Phosphorylation or mutations in these regions, can alter their interactions with the core of the protein, thereby affecting nucleotide binding at NBD1 as well as the equilibrium between different NBD1 conformations that ultimately modulate the activity of the K ATP channel. iii Acknowledgments There are a number of people who have truly enhanced my graduate experience over the past years. My deepest gratitude is to my supervisor Prof. Voula Kanelis who has always been supportive and has helped me grow as a well-rounded researcher. I am grateful to her mentorship and unwavering support and enthusiasm, and she never misses an opportunity to turn something into a teachable moment. I am grateful to my committee members, Prof. R. Scott Prosser and Prof. Drew Woolley, for their expertise, insightful suggestions and technical support over the years. Prof. Scott Prosser is an avid researcher and his enthusiasm helped instill my interest in biophysics during my undergraduate years. Prof. Drew Woolley is very patient and accommodating and has taught me many valuable lessons in biochemistry and chemical biology. I would also like to thank Prof. Ulrich Krull, Prof. Mark Nitz and Prof. Xiao-an Zhang for helping shape my understanding of biochemistry and NMR. I would like to acknowledge Prof. Brian Shilton, Prof. Jumi Shin and Prof. Deborah Zamble for reviewing my thesis. I would also like to acknowledge Dr. John Rubinstein for all of his suggestions and advice on all of our publications and Prof. Barry Green for his support. I am also appreciative for the support of my several lab mates over the past years with special thanks to Dr. Jorge P. Lopez-Alonso, Marijana Staglijar, Lynn K. Ikeda, Clarissa R. Sooklal, Serisha Moodley, Sasha Weiditch and Alexandria Albanese. My past and present colleagues have been instrumental to create an enjoyable and engaging working environment. I would also like to acknowledge my colleagues from other labs who have all helped me in various ways and have provided me with a remarkable graduate experience throughout the years. I am grateful to the support from the many past and present members of the Prosser Lab including Dr. Sameer Al-Abdul-Wahid, Dr. Rohan Alvares, Dr. Libin Ye, Tae Hun Kim and Joshua Hoang. I would also like to thank Dr. Sacha Larda from the Prosser Lab for his leadership during our time on the CPS graduate student union. I am grateful to my colleagues from the Shin Lab (Dr. Sam Sathiamoorthy, Dr. Pam Nge, Dr. Ichiro Inamoto, Dr. Inder Sheroan, Dr. Antonia DeJong and Alexandra Strak) for their support. I am also thankful to the support from the MacDonald Lab iv (Dr. Quasim Saleem and Angel Lai), Krull Lab (Uvaraj Uddaysankar), Kay lab (Dr. Ranjith Muhandiram) and the Stewart Lab (Dr. Colin De Mill, Marzena Serwin). I would especially like to thank Prof. Peter Macdonald for providing me multiple opportunities to grow as an educator and teach organic chemistry. I would also like to extend my appreciation to the many members of the Chemical and Physical Sciences Department for their expertise and assistance while I was teaching, including Prof. Jumi Shin, Prof. Patrick Gunning, Prof. Juris Strautmanis, Dr. Sreekumari Nair, Dr. Krish Radhakrishna, Liz Kobluk, Angela Sidoriak, Rubina Lewis, Heidi Moore, and Donna Coulson. I am grateful to doctoral grants from the Canadian Institutes of Health Research and Queen Elizabeth II Science and Technology. Finally, I would especially like to thank my parents and my sister for their unwavering support throughout my graduate studies. v Table of Contents Acknowledgments ........................................................................................................................................................ iv Table of Contents ......................................................................................................................................................... vi List of Figures ............................................................................................................................................................... x List of Tables .............................................................................................................................................................. xiii List of Equations......................................................................................................................................................... xiv List of Abbreviations ................................................................................................................................................... xv Chapter 1 Introduction ............................................................................................................................................... 1 1 Overview of K ATP channels ...................................................................................................................................... 1 1.1 Molecular Architecture of K ATP channels........................................................................................................ 1 1.2 Overview of the Kir subunit ............................................................................................................................ 2 1.3 Overview of the SUR subunit ......................................................................................................................... 4 1.3.1 Overview of the ABC transporters .................................................................................................... 4 1.3.2 General Structure of ABC transporters ............................................................................................. 5 1.3.2.1 Arrangement of domains in ABC proteins ...................................................................... 6 1.3.2.2 Subdivisions in the ABCC subfamily .............................................................................. 8 1.3.2.3 Structure of the Membrane Spanning Domains (MSDs) ................................................. 8 1.3.2.3.1 The role of MSD0 and the L0 linker ................................................................................ 9 1.3.2.4 Structure of the Nucleotide Binding Domains (NBDs) ................................................. 11 1.3.3 SUR isoforms and splice variations ................................................................................................ 13 1.4 Nucleotide regulation of K ATP channel activity and kinetics ......................................................................... 15 1.4.1 Phosphotransfer Networks .............................................................................................................. 18 1.5 Physiological Role of K ATP channels ............................................................................................................. 20 1.6 Additional Regulation of K ATP channels ....................................................................................................... 22 1.6.1 Pharmaceutical Regulation ............................................................................................................. 22 1.6.1.1 Sulfonylureas ................................................................................................................. 22 1.6.1.2 Potassium