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Role of Coupled Dynamics and a Strictly Conserved Lysine Residue in the Function

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Role of Coupled Dynamics and a Strictly Conserved Lysine Residue in the Function Role of Coupled Dynamics and a Strictly Conserved Lysine Residue in the Function of Bacterial Prolyl-tRNA Synthetase and Substrate Binding by a Related trans- Editing Enzyme ProXp-ala Dissertation Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Brianne Leigh Sanford, B.S. Graduate Program in Chemistry The Ohio State University 2014 Dissertation Committee: Dr. Karin Musier-Forsyth, Advisor Dr. Mark Foster Dr. Jane Jackman i Copyright by Brianne Leigh Sanford 2014 ii Abstract During protein synthesis, aminoacyl-tRNA synthetases (ARSs) are responsible for activating a specific amino acid and charging it to its cognate tRNA. Amino acids are often misactivated and mischarged by ARSs due to their similar size. To increase accuracy, about half of ARSs have evolved editing mechanisms. Prolyl-tRNA synthetase (ProRS) requires editing to distinguish Ala and Cys from cognate Pro. In many bacterial species, this is accomplished through a triple-sieve editing mechanism. In the first sieve, larger amino acids are excluded while Ala, Cys and Pro are all activated and charged to tRNAPro. In the second sieve, the insertion (INS) domain accepts Ala-tRNAPro for deacylation. Finally, in the third sieve Cys-tRNAPro is accepted and deacylated by YbaK, an editing protein that is homologous to the INS domain. ProRS is a multidomain enzyme with a catalytic domain, anticodon binding domain, and the INS domain. The internal dynamics of this multidomain protein were probed to test the hypothesis that functional dynamics of the domains are disrupted upon deletion of the INS domain, resulting in decreased catalytic activity. We show that the INS and active site domains show anticorrelated dynamics. The dynamics of the INS domain and a catalytically important proline-binding loop are also coupled. Mutation of conserved residues at the interface of the INS and catalytic domains exhibited a significant effect on the dynamics of the proline-binding loop and ultimately catalytic function of the enzyme. ii In Escherichia coli (Ec) ProRS these domain dynamics are propagated through a set of dynamically coupled residues that form a pathway of residue-to-residue interactions between the catalytic and editing domains. Residues in this contiguous network are generally highly conserved and mutation along these pathways results in a significant impact on enzyme function. The role of a strictly conserved Lys residue in the INS active site in Ala-tRNAPro binding and catalysis was also examined. This Lys at position 279 in Ec ProRS was calculated to have a perturbed pKa of 14.2. The protonated state of K279 is stabilized by a nearby acidic residue. Mutation of this acidic residue to a positively charged residue resulted in increased deacylation activity. When a mutant was made wherein the positions of the conserved Lys and charged residue were swapped, deacylation activity was abolished. This supports the conclusion that both the positive charge and position of the Lys are critical for proper substrate orientation. There exists a modified triple sieve editing mechanism in Caulobacter crescentus (Cc), which encodes for a ProRS lacking a full-length, functional INS domain. A small free-standing protein, ProXp-ala, is homologous to the INS domain and deacylates mischarged Ala-tRNAPro. Cc also encodes for a YbaK to deacylate Cys-tRNAPro. ProXp- ala prefers to deacylate Ala-tRNAPro based on recognition of C1:G72 and A73 in the acceptor stem. NMR studies will define the tRNA acceptor stem interaction sites on ProXp-ala, shedding light on which elements in the enzyme define the Ala-tRNA substrate specificity. Altogether, the work presented here reveals new insights into the dynamics of aminoacyl-tRNA synthetases and how they interact with their substrates. iii Dedication This is dedicated to my loving husband and my ever-supportive parents and family. iv Acknowledgements First and foremost, I would like to thank my advisor Dr. Karin Musier-Forsyth for providing me with the opportunity to join her lab and work on a range of projects. She has set up a great research environment where I had access to a diverse set of instruments and techniques required to accomplish the goals of my projects. Under her guidance, she has pushed me to become an independent thinker and has taught me to always think about the “big picture”–how do my everyday experiments contribute to the goal of the project? I believe it is these skills, which are not necessarily taught in every research lab, are what make a great scientist. I would also like to thank my collaborators with whom I have worked together on great projects. Dr. Sanchita Hati sparked my interest in studying aminoacyl-tRNA synthetases during my undergraduate studies, and I was fortunate to continue these projects in graduate school in Dr. Musier-Forsyth’s lab. I am also grateful for the wonderful collaboration with Dr. Mark Foster’s lab. Dr. Foster is very good at pushing me to think about my projects from another point of view, helping me think of other ways to approach a problem. Through this collaboration, I have had the opportunity to work with a brilliant graduate student Eric Danhart. He has very patiently answered my many questions about the NMR data collected for my projects. I would also like to thank Dr. Jane Jackman for helping me think critically and prepare for my candidacy exam and for helping me complete my final thesis. v I also owe a great debt of gratitude to all the members of the Musier-Forsyth lab who have helped me think critically about my projects and even everyday questions about techniques in lab. I have had many brainstorming sessions with Oscar Vargas- Rodriguez, Jo Marie Bacumso and Drs. Mom Das and Marina Bakhtina. I am very appreciative for frequent discussions about computational methods and RNA structures with Dr. William Cantara. I also have several friends to thank for keeping me sane and focused throughout graduate school– Tiffiny Rye-McCurdy and Alice Duchon for our frequent coffee breaks and my two friends Drs. Tricia Meyer and Allyson Fry with whom I started this whole journey. Lastly, I have to thank my family members that have always supported my endeavors. My husband has been by my side during every leg of this journey, celebrating my accomplishments and at times, providing much needed encouragement. My parents and sisters have also been very supportive, despite the many miles between us. I also have to thank my aunt for her constant motivation and my husband’s parents for their kind support. Without all these people in my life and at Ohio State, I would not have been able to start or finish this journey and for that I am grateful. vi Vita 2008………….............................................. B.S. Chemistry, cum laude University of Wisconsin – Eau Claire 2008-present……………………………….. Graduate Researcher Assistant, Department of Chemistry and Biochemistry, The Ohio State University Publications 1. Weimer K, Shane B, Brunetto M, Bhattacharyya S, and Hati S. Evolutionary Basis for the Coupled-domain Motions in Thermus thermophilus Leucyl-tRNA Synthetase. (2009) J. Biol. Chem., 284:10088-10099. 2. Sanford B, Cao B, Johnson J, Zimmerman K, Strom A, Mueller R, Bhattacharyya S, Musier-Forsyth K, and Hati S. Role of Coupled Dynamics in the Catalytic Activity of Prokaryotic-like Prolyl-tRNA Synthetases. (2012) Biochemistry. 51 (10), 2146-2156. 3. Johnson J, Sanford B, Strom A, Tadayon S, Lehman B, Zirbes A, Bhattacharyya S, Musier-Forsyth K, and Hati S. Multiple Pathways Promote Dynamical Coupling Between Catalytic Domains in Escherichia coli Prolyl-tRNA Synthetase. (2013) Biochemistry. 52 (25), 4399-4412. 4. Bartholow T, Sanford B, Cao B, Schmit H, Johnson J, Meitzner J, Bhattacharyya S, Musier-Forsyth K, and Hati S. Strictly Conserved Lysine of Prolyl-tRNA Synthetase vii Editing Domain Facilitates Binding and Positioning of Misacylated tRNAPro. (2014) Biochemistry. 53 (6), 1059-1068. Fields of Study Major Field: Chemistry viii Table of Contents Abstract………………………………………………………………………………….. ii Dedication……………………………………………………………………………..... iv Acknowledgements…………………………………………………………………...…. v Vita……………………………………………………………………………………... vii Publications…………………………………………………………………………….. vii List of Tables…………………………………………………………………………… xv List of Figures………………………………………………………………………….. xvi List of Schemes............................................................................................................... xix List of Symbols and Abbreviations…………………………………………………..... xx Main Chapters: 1. Introduction………………………………………………………………………...... 1 1.1. Background…………………………………………………………………….. 1 1.1.1. The Central Dogma of Molecular Biology………………………………. 1 1.1.2. Aminoacyl-tRNA Synthetases………………………………………….... 2 1.1.3. Transfer RNA structure and aminoacyl-tRNA synthetase recognition……………………………………………………………….. 4 1.2. Quality Control During Protein Synthesis....………………………………….. 5 1.2.1. Pre-transfer Editing……………………………………………………… 6 1.2.2. Post-transfer Editing……………………………………………………... 7 ix 1.2.2.1. Trans-editing factors…………………………………............... 8 1.2.2.2. Post-transfer editing in trans by the INS superfamily……...….. 9 1.3. Significance of editing in vivo……………………………………………......... 10 1.4. Purpose of this study…………………………………………………………... 11 2. Role of Coupled Dynamics in the Catalytic Activity of Prokaryotic-like Prolyl-tRNA Synthetases………………………………………………………………………….. 19 2.1. Introduction……………………………………………………………………. 19 2.2. Materials and Methods…………………………………………………………
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