MOLECULAR INTERACTIONS AND DYNAMICS IN CYCLIC AMP SIGNALING BALAKRISHNAN SHENBAGA MOORTHY NATIONAL UNIVERSITY OF SINGAPORE 2011 MOLECULAR INTERACTIONS AND DYNAMICS IN CYCLIC AMP SIGNALING BALAKRISHNAN SHENBAGA MOORTHY (Master of Technology in Biotechnology) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS Completing my doctor of philosophy (PhD) is possible only because of the continuous support I received throughout my graduate career from many people. In addition to my friends and family members, I would like to thank the following people who supported in all the aspect of my personal and career life. I would like to express my sincere thanks to my project supervisor Dr. Ganesh Srinivasan Anand. I am proud to address myself as his first PhD graduate student. I express my deep sense of gratitude for his continuous guidance, timely advice, discussions and support through all the stages of my project. He also taught me in solving various problems encountered during my project. His passion for science provided me encouragement to successfully complete this project and to become a scientist. I am very much grateful to National University of Singapore for providing me the environment, facilities and full support to carry out my graduate study. I would like to extend my thanks to my PhD qualifying examiners, Prof. Liou Yih Cherng, Prof. Sanjay Swarup and Prof. Naweed Naqvi for their invaluable advices during discussions. I thank Prof. Ivana Mihalek, Bioinformatics Institute, Singapore and Prof. Giuseppe Melacini from McMaster‟s University, Hamilton for their current and future collaborations on my project. I would also like to thank Prof. Susan S. Taylor, Prof. William Loomis from University of California, San Diego and Prof. Linda Kenney from Mechanobiology Institute, Singapore for sharing clones and reagents for our studies. I would like to thank Prof. K Swaminathan and Prof. J Sivaraman for their scientific ideas in encouraging me to extend my project for crystallographic studies. i I thank our lab post-doc Dr. Gao Yunfeng for her help in molecular cloning and initial support in learning lab safety procedures. I appreciate my labmates, Suguna Badireddy, Tanushree Bishnoi, Srinath Krishnamurthy, Wang Loo Chien, Anusha Vedagiri, Jane Lin Liqin, Aparna Sankararaman, Christina Yap Xiaojun, Liang Yuan Yuan for their useful discussions and friendship. I thank Mr. Lim Teck Kwang for his technical support with mass spectrometry. I take this opportunity to thank my roommates, Raghu, Jayaraj, Thanneer, Kiran, Lakshmi, Vamsi and Prashant for their help and support in Singapore. I like to specially thank Dr. B. C. Karthik for his useful discussions and advice during lunch and tea sessions. I declare my thanks to my friends here in Singapore and in India for their continuous encouragements and help throughout my graduate career. I should thank my family members Amma, Appa, Sisters, Athai and Maama for their love and affection on me and making my life colorful. Last but not least, I wish to thank my wife Poornima for her love and continuous support during difficult situations. I thank God ever for giving me such a caring and understanding better half. ii TABLE OF CONTENTS PAGE NO ACKNOWLEDGEMENT i TABLE OF CONTENTS iii LIST OF ABBREVIATIONS viii SUMMARY x LIST OF TABLES xiii LIST OF FIGURES xiv LIST OF PUBLICATIONS xxv INTRODUCTION CHAPTER 1: Cooperativity and allostery in cAMP-dependent activation of Protein Kinase A: Monitoring conformations of intermediates by amide hydrogen/deuterium exchange 1.1 Introduction 16 1.2 Materials and Methods 1.2.1 Reagents 20 1.2.2 Purification of RIα(92-379)(R209K) and C- subunit 21 1.2.3 Amide HDXMS 21 1.3 Results and Discussion 1.3.1 Pepsin digestion of RIα(92-379)R209K and C- subunit 24 1.3.2 Evidence that cAMP binding to RIα(92-379)R209K:C holoenzyme does not lead to dissociation of the complex 31 1.3.3 cAMP binding to RIα(92-379) R209K:C holoenzyme decreases deuterium exchange in PBC:B 31 1.3.4 cAMP binding to CNB-B increases deuterium exchange 33 iii at interface between CNB-B and C-subunit 1.3.5 Effects of cAMP binding to RIα(92-379)R209K:C holoenzyme: Changes in PBC:A of RIα 34 1.3.6 Global conformational changes in RIα 35 1.3.6.1. Pseudosubstrate region 35 1.3.6.2. αB/C:Α, αC‟:A and αA:B helix 35 1.4 Conclusion 38 CHAPTER 2: Phosphodiesterases Catalyze Hydrolysis of cAMP-bound to Regulatory Subunit of Protein Kinase A and Mediate Signal Termination 2.1 Introduction 41 2.2 Materials and Methods 2.2.1 Materials 45 2.2.2 Cloning and Expression of a C-terminal deletion domain mutant of RegA 46 2.2.3 Protein Expression and Purification 47 2.2.4 Pull-Down Assays 47 2.2.5 Fluorescence Spectroscopy 48 2.2.6 Phosphodiesterase Assay 49 2.2.7 Fluorescence Polarization (FP) Assay for cAMP Dissociation 51 2.2.8 Amide HDXMS - (LC-ESI-QTOF-MS) 52 2.2.9 Amide HDXMS - (MALDI-TOF-MS) 54 iv 2.3 Results 2.3.1 Deletion Mutagenesis Indicates that the Catalytic Domain of RegA Mediates AKAP-independent Interactions with the CNB:A Domain 56 2.3.2 Measurement of Binding Affinity of RIα to RegA by Fluorescence Quenching 59 2.3.3 RIα Binding Induces a 13X Increase in RegA Phosphodiesterase Activity 60 2.3.4 Mapping RIα-RegA Interactions by Amide HDXMS 63 2.3.5 Three Regions on RIα showed Decreased Solvent Accessibility in the RegA-RIα Complex: Phosphate Binding Cassette, β-stands 1-2 and a:B-C-helices 69 2.3.6 RegA Catalyzes Hydrolysis of cAMP-bound to RIα 71 2.4 Discussion 2.4.1 RegA Phosphodiesterase is capable of Hydrolyzing cAMP-bound to RIα 77 2.4.2 Dual Function of RIα as Inhibitor of C-subunit and Activator of PDEs 80 2.4.3 RIα Mediates Distinct but Overlapping Interactions with PKA C- subunit and RegA-PDE 80 CHAPTER 3: Basis for the activation of phosphodiesterase in RegA-RIα interactions 3.1 Introduction 3.2 Materials and Methods 3.2.1 Materials 85 v 3.2.2. Protein Expression and Purification 85 3.2.3. Amide HDXMS 86 3.2.4. Pull down assay with immobilized cAMP -bound RIα(91-244 87 3.3 Results 3.3.1. Peptide Array Analysis for RegA:RIα Interactions 88 3.3.2. Pepsin digestion and peptide identification for RegA 91 3.3.3. Amide HDXMS 92 3.3.4. RegA-Rα interactions alter regions within metal binding site 94 3.3.5. Substrate binding pocket is stabilized during RegA- RIα interactions 95 3.3.6. RegA primes RIα for reassociation with C-subunit 98 3.4 Discussion 99 3.5 Conclusion 101 CHAPTER 4: Multi-State Allostery in Response Regulators: Phosphorylation and Mutagenesis Activate RegA via Alternate Modes 4.1 Introduction 106 4.2 Materials and Methods 4.2.1 Reagents 110 4.2.2 Cloning, expression and purification of RegA and mutants 110 vi 4.2.3 Phosphodiesterase activity assay 111 4.2.4 Amide HDXMS studies 111 4.3 Results 4.3.1 Mutation of the aromatic switch residue enhances phosphodiesterase catalysis of RegA 114 4.3.2 Phosphorylation causes decreased exchange across the Receiver Domain which reflects large scale stabilization and reduction in backbone dynamics 116 4.3.3 Interdomain linker and catalytic loop residues also show decreased deuterium exchange upon phosphorylation 119 4.3.4 Receiver domain of activating mutant (RegA F262W) is more dynamic compared to phosphorylated as well as unphosphorylated RegA 120 4.3.5 Deuterium exchange of the linker and catalytic domains in activating mutant, RegA F262W are distinct but overlap with phosphorylated RegA 120 4.3.6. Receiver domain decreases deuterium exchange within the catalytic PDE domain without altering activity 121 4.4 Discussion 4.4.1 Allosteric coupling of phosphorylation and aromatic switch residue 131 4.4.2 Dynamics of the catalytic domain in phosphorylated RegA and RegA F262W are overlapping yet distinct 132 4.4.3 Phosphorylation-dependent activation of RegA through decreases in protein-wide dynamics 133 4.5 Conclusion 139 FUTURE DIRECTIONS REFERENCES 142 146 vii LIST OF ABBREVIATIONS AKAP: A- Kinase anchoring protein AC: Adenylyl cyclase BME: β- Mercaptoethanol C subunit: Catalytic subunit of PKA cAMP: Cyclic adenosine 3‟, 5‟- monophosphate CIAP: Calf intestinal alkaline phosphatase CNB-A and CNB-B: cyclic nucleotide binding domain A and B respectively FM: Fluorescein maleimide GST: Glutathione S-Transferase FP: fluorescence polarization LC-ESI QTOF: Liquid chromatography- Electrospray ionization Quadrupole Time-of-flight MALDI-TOF: Matrix-Assisted Laser Desorption Ionization Time-of-Flight NHS: N-hydroxysuccinimide PCR: polymerase chain reaction PDE: cyclic nucleotide phosphodiesterase PKA: Protein kinase A R-subunit: Regulatory subunit of PKA viii HDXMS studies with truncated mutant RIα (91-379) having two cAMP binding domain and RIα full length (1-379) having two cAMP binding domain, inhibitory region and the dimerization domain. Here we believe that physiological RIα will mediate multivalent interactions with RegA leading to higher binding affinities than observed with RIα(91244). This will provide invaluable insight into the structure and conformational dynamics of this seminal macromolecular complex in cAMP Signaling. Also studying unphosphorylated/phosphorylated full length RegA interaction with RIα may provide mechanism that link the two-component system and the cAMP Signaling in Dictyostelium discoideum. 2. Site directed mutational analysis to confirm the binding sites From our previous amide HDXMS results, it is very clear that RegA catalytic domain binds specifically to the cAMP binding A domain of RIα. Residues from RIα and RegA critical for interactions can be mutated to Ala to test and confirm the binding of RegA to RIα. Mutants of RIα which are critical for interactions with RegA can be tested for their ability to interact with and inhibit the C-subunit of PKA. This will reveal the mechanism of regulation of RIα-RegA interactions by the C-subunit and signal termination. Studies on point mutants of RegA that affect RIα-RegA interactions and point mutants that affect hydrolysis of cAMP will allow us to differentiate binding from enzyme activation mechanism. Identifying residues from RegA critical for interactions with RIα will help us to narrow down the process in identifying the mammalian homolog. 143 3. Computational modelling of the RegA-RIα interface We are collaborating with computational biologist to map the interaction surface of RegA with RIα by mapping surfaces that show decreased solvent accessibility upon complex formation using a combination of computational docking and amide HDXMS (Anand et al., 2003). Results from HDX can be combined with computational modelling techniques with the help of our collaborator, Dr. Ivana Mihalek, Bioinformatics Institute (BII), Singapore. 4. Monitoring interactions inside the cell Monitoring RIα mediated activation of PDEs inside the cell may lead to the finding of novel biological roles for this interactions. Cellular cAMP and PKA FRET reporter assays will be highly efficient in the activation studies. The rIa gene can be cotransfected into cells along with cytosolic and organelle-directed cAMP or PKA reporters to track the subcellular localization of separate PDE isoforms. Common cell lines such as HeLa can be used to monitor the activation of PDEs by RIα. HeLa cell lines can be subjected to siRNA knockdown of the RIα gene. The effects of decreased RIα expression on PDE activity can be monitored and the kinetics of cAMP hydrolysis can be compared between the wild-type cells and RIα knocked down cell lines. 5. Structure determination of RIα-RegA complex Structures of different states of deletion mutants of cAMP-free RIα, cAMP-bound RIα and in complex with the C-subunit have been previously solved by crystallography. A high-resolution structure of the RegA-RIα complex in combination with biophysical methods would be invaluable in mapping the magnitude of conformational changes 144 within RIα upon complexation with RegA, This would also provide insights into the mechanism by which RIα functions as an activator for PDE catalysis. This would further test our hypothesis that RIα-RegA interactions stabilize the substrate binding pocket and the dimer form of RegA to increase the PDE activity of RegA. Now, we are in process of screening conditions for the crystallization of RIα-RegA complex. The outcomes from our present research have provided molecular details of a regulatory mechanism in an important Signaling pathway. Given the physiological and pharmacological importance of both PKA and PDE, these findings will greatly drive research in this field. 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Bacteriol., 178, 4208-4215. 158 [...]... connected to two cAMP binding domains by a variable, disordered linker region The proximal cAMPbinding domain is referred to as the cAMP binding A domain and the distal domain is the cAMP binding B domain The C-subunit on the other hand is a globular protein and can be shown to consist of an N and C-terminal lobe and enclosing an ATP and substratebinding cleft The R-subunit consists of an N-terminal dimerization... cooperative and sequential with cAMP binding first to CNB-B and then to CNB-A Mutation of Arg 209 to a Lys in CNB-Α of R- subunit abolishes high-affinity cAMP binding to the CNB-Α without significantly affecting binding of cAMP to CNB-B The holoenzyme, RIα(92379)R209K:C provides an ideal model system to probe the effects of a single cAMP binding to CNB-B and studying effects of a single cAMP bound intermediate... an N-terminal dimerization domain followed by pseudosubstrate or the inhibitor motif to which the catalytic core of C subunit interacts The R-subunit lacking the N-terminal dimerization domain, RIα(92-379) and RIα(91244), retains high affinity binding to the C-subunit and provides a minimal monomeric model for examining R-C interactions as well as cAMP binding to CNB-A and CNB-B (Kim et al., 2007; Su... subunits and the RIαC holoenzyme (Kim et al., 2005; Taylor et al., 2007) show snapshots of PKA in its two stable end states (inactive and active) (Figure: ii) So, understanding the mechanism of activation and the intermediates in PKA is highly important in cAMP Signaling For activating this enzyme one molecule of cAMP has to bind to each of the cAMP binding domains, this is assumed to occur in a serial... regulatory domains (Figure: iii) for instance, calmodulin 5 binding domain, GAF (cGMP specific PDE, Adenylyl cyclase, and Fh1A) domain, UCR (Upstream Conserved Region) domain, PAS (Period clock protein, Aryl hydrocarbon receptor nuclear translocator, and Single minded protein) domain, REC (receiver) domain and Transmembrane domain (Conti and Beavo, 2007; Francis et al., 2011) Figure iii: Domain organization... serial manner First, one molecule of cAMP binds to the B domain (CNB-B) which cooperatively facilitates binding of a second molecule of cAMP binding to the A domain (CNB-A) leading to the release of the catalytic 4 subunit The cAMP binding domain B is believed to act as “gatekeeper” for modulating cAMP access to domain A (Taylor et al., 2007) The role of B domain and the mechanism by which it controls... This 3 information indicates that RIα is uniquely required for effective regulation of the PKA kinase and stresses the importance of RIα localization, regulation and molecular interactions iv Tightly regulated PKA activation - Role of two cAMP binding domains The mammalian RIα isoform is modular and extended proteins having a very similar domain organization with an N-terminal dimerization domain connected... both CNB-A and CNB-B In PBC:A, the critical conserved residues Arg 209 and Glu 200 and in PBC:B, Arg 333 and Glu 324 anchor the cyclic phosphate and 2‟OH moieties of cAMP (yellow) respectively 19 1.2 Cartoon showing step-wise cAMP-mediated activation of PKA (Rsubunit in red, C-subunit in blue, *- represents a molecule of cAMP, X- represents mutation that abolishes high-affinity binding of cAMP) Activation... role of cAMP in mammalian cells is mediated through the activation of cAMP dependent Protein Kinase A (PKA) cAMP binding induces large conformational changes within the R-subunit leading to dissociation of the active C-subunit Although crystal structures of end-point, inactive and active states are available, the molecular basis for cooperativity in cAMP-dependent activation of PKA is not clear In this... increased exchange upon binding cAMP are in red and suggest disruption of the specific intersubunit contacts mediated by the CNB-B domain with the C-subunit (yellow arrow), (site 4 of R-C intersubunit interactions, (Kim et al., 2007)) 33 1.7 Increased exchange upon binding of a single molecule of cAMP to RIα(92-379) R209K:C holoenzyme, within residues 230-270 (spanning α:B/C and α:A of CNB-B) region . MOLECULAR INTERACTIONS AND DYNAMICS IN CYCLIC AMP SIGNALING BALAKRISHNAN SHENBAGA MOORTHY NATIONAL UNIVERSITY OF SINGAPORE 2011 MOLECULAR INTERACTIONS AND. Domain organization of RIα showing an N-terminal dimerization/docking domain (D/D) (gray hashed box) connected by a linker to two tandem cAMP-binding domains, CNB-A and CNB-B in green. The linker. critical for RegA-RIα interactions include the metal binding M site and substrate binding Q pocket in RegA. Results from the pull down experiment show that RegA binding primes cAMP-bound RIα for