Defining the contours of cyclic nucleotide mediated regulatory switches from Prokaryotes to Eukaryotes Suguna Badireddy M. Sc (Biochemistry) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgement Being at the juncture of completing my doctor of philosophy (Ph.D), I am extremely elated for accomplishing this professional milestone, which was my college dream. This became possible only because of continuous guidance, support, caress and affection of significant few who surrounded my life in this journey and added success and joy in their own flavors. On that note, I wish to extend my heartfelt gratitude to my family, friends, teachers and colleagues who continue to help me in ways that I can never thank them. If accomplishment had to be measured as distance between one’s origins and one’s final achievement, then I am indebted to my supervisor Dr. Ganesh Anand, for not only building in me enough confidence to start my work in an area that was completely foreign to me but also helping me prune my skills continuously and setting targets for the tasks which made me work an extra inch every time, resulting in completion of my doctorate in time. I am also extremely thankful to my co-supervisor Dr. K. Swaminathan for not only his knowledge and time in helping me in crystallographic studies but also for his optimistic and enthusiastic words in tough times. I would like to extend my thanks to my Ph.D qualifying examiners Prof. Sivaraman J, Prof. Ng. Davis and Prof. Kim chu-young for their invaluable advices during discussions. I would also like to thank Prof. Susan S.Taylor, university of California, San Diego for sharing clones for our studies. I thank my lab mates B.S. Moorthy, B. Tanushree, Jeremy and K. Srinath for their useful discussions, support, friendship and fun on and off the work. I am extremely thankful to Nilofer Husain, my i friend since undergraduate days in India for being my closest pal and sharing my blues and smiles on both personal and professional fronts during my stay here. I would not have achieved this milestone with out the support of my family and am thankful to my mom, uncle, aunt and in-laws. But for their continuous words of support and enthusiasm through out my work here, I would not have been comfortable enough to complete on time. I am thankful to god for giving me a loving husband and my best friend who was always there for me in whatever I and this work is not just mine but ours together. ii Table of Contents Page No Acknowledgement i Table of contents iii Summary ix List of Tables xiii List of Figures xiv List of abbreviations xx List of publications xxii General Introduction CHAPTER 1: Cyclic AMP analog blocks kinase activation by stabilizing inactive conformation: Conformational selection highlights a new concept in allosteric inhibitor design 1.1 Introduction 22 1.2 Materials and Methods 26 1.2.1 Reagents 26 1.2.2 Expression and purification of PKA C-subunit 26 1.2.3 Expression and purification of PKA RA 27 iii 1.2.4 Purification of PKA holoenzyme 28 1.2.5 Crystallization, data collection, structure solution 28 and refinement, of apo RA and cAMP-bound RA 1.2.6 Crystallization, data collection, structure solution, 29 and refinement of Rp-bound PKA RA 1.2.7 Amide hydrogen/deuterium (H/D) exchange mass 32 spectrometry 1.2.8 Gas phase protein structure measurement by ion 35 mobility mass spectrometry 1.3 Results 36 1.3.1 Structures of apo and cAMP-bound RA 36 1.3.2 Structure of RA bound to Rp 39 1.3.3 Structural differences between Rp-bound RA 42 and apo, cAMP- and C-subunit-bound states 1.3.4 1.3.3.1. !-subdomain 42 1.3.3.2. "-subdomain 43 Amide hydrogen/deuterium (H/D) exchange 44 mass spectrometry analysis 1.3.5 1.4 Ion mobility mass spectrometry 50 Discussion 52 1.4.1 52 Conformational selection in the R-subunit: Rp stabilizes inactive H-conformation iv 1.4.2 Mechanism of cAMP action and basis for 55 antagonism of Rp 1.4.3 Identification of highly selective allosteric inhibitors 58 that specifically bind and stabilize 'inactive' conformations CHAPTER 2: Cooperativity and allostery in cAMP-dependent activation of Protein Kinase A: Monitoring conformations of intermediates by amide hydrogen/deuterium exchange 2.1 Introduction 62 2.2 Materials and Methods 66 2.2.1 Reagents 66 2.2.2 Purification of RI"(92-379)(R209K) and C- subunit 66 2.2.3 Amide hydrogen/deuterium (H/D) exchange mass spectrometry 67 2.3 Results and Discussion 69 2.3.1 Pepsin digestion of RI"(92-379)R209K and C- subunit 70 2.3.2 Evidence that cAMP binding to RI"(92-379)R209K:C 75 holoenzyme does not lead to dissociation of the complex 2.3.3 cAMP binding to RI"(92-379) R209K:C holoenzyme 75 decreases deuterium exchange in PBC:B 2.3.4 Effects of cAMP binding to RI"(92-379)R209K:C 77 holoenzyme: Changes in PBC:A of RI" 2.3.5 cAMP binding to CNB-B increases deuterium 77 exchange at interface between CNB-B and C-subunit 2.3.6 Global conformational changes in RI" 79 v 2.4 2.3.6.1. Pseudosubstrate region 79 2.3.6.2. "B/C:#, "C`:A and "A:B helix 79 Conclusion 82 CHAPTER 3: Cyclic AMP-induced Acetyltransferases Conformational Changes in Mycobacterial 3.1 Introduction 85 3.2 Materials and Methods 89 3.2.1 Reagents 89 3.2.2 Cloning and Mutagenesis 89 3.2.3 Expression, purification and characterization 90 protein of proteins 3.2.4 In vitro BRET assays 90 3.2.5 In vitro acetylation assays 91 3.2.6 Amide hydrogen/deuterium (H/D) exchanges mass 92 spectrometry 3.3 Results 94 3.3.1 94 Conformational changes in KATms monitored by BRET 3.3.2 Cyclic AMP binding induces large conformational 96 throughout the CNB domain 3.3.3 Amide hydrogen/deuterium (H/D) exchanges mass 97 spectrometry analysis vi 3.3.4 Differential effects of the cAMP analogs 105 8Br-sp-cAMPS and 8Br-Rp-cAMPS 3.3.5 Linker region is important for propagating cAMP 107 induced conformational changes in KATms 3.3.6 Mutation in the linker region abolish cAMP- 108 mediated activation of AT activity 3.3.7 linker –mediated conformational changes in the 111 presence of cAMP are conserved in Rvo998 3.4 Discussion 113 CHAPTER 4: Distinct modes of binding and conformational changes induced by cAMP and cGMP in the isolated GAF-B domain of Anabaena adenylyl cyclase, CyaB2 4.1 Introduction 121 4.2 Materials and Methods 124 4.2.1 124 Reagents 4.2.2. Expression and Purification of N-terminal 125 hexahistidine tagged GAF-B domain 4.2.2. Amide hydrogen/deuterium exchange mass 125 spectrometry of GAF-B 4.3 4.4 Results 128 4.3.1. cAMP mediated changes in GAF-B domain 128 4.3.2. Cyclic GMP mediated changes in GAF-B domain 133 4.3.3. Sp and Rp mediated changes in GAF-B domain 138 Discussion 139 vii 4.4.1 Ligand mediated conformational changes 4.4.2. Importance of equatorial and axial oxygens 140 142 Conclusion 144 Future directions 148 References 150 viii Summary The regulatory (R) subunit of Protein Kinase A (PKA) serves to modulate the activity of PKA in a cAMP-dependent manner and exists in two distinct and structurally dissimilar, endpoint cAMP-bound 'B' and C-subunit-bound 'H'-conformations. Here we report mechanistic details of cAMP action as yet unknown through a unique approach combining X-ray crystallography with structural proteomics approaches- amide hydrogen/deuterium exchange and ion mobility mass spectrometry, applied to the study of a stereospecific cAMP phosphorothioate analog and antagonist((Rp)-cAMPS). X-ray crystallography shows cAMP-bound R-subunit in the ‘B’ form but surprisingly the antagonist Rp-cAMPS-bound R-subunit crystallized in the ‘H’ conformation which was previously assumed to be induced only by C-subunit-binding. Apo R-subunit crystallized in the ‘B’ form as well but HDX mass spectrometry showed large differences between apo, agonist and antagonist-bound states of the R-subunit. Further ion mobility reveals the apo R-subunit as an ensemble of multiple conformations with collisional cross-sectional areas (CCS) spanning both the agonist- and antagonist-bound states. Thus contrary to earlier studies which explained the basis for cAMP action through 'induced fit' alone, we report evidence for conformational selection, where the ligand-free apo form of the Rsubunit exists as an ensemble of both 'B' and 'H' conformations. While cAMP preferentially binds the 'B' conformation, Rp-cAMPS interestingly binds the 'H' conformation. This reveals the unique importance of the equatorial oxygen of the cyclic phosphate in mediating conformational transitions from 'H' to 'B' forms highlighting a novel approach for rational structure-based drug design. Ideal inhibitors such as RpcAMPS are those that preferentially 'select' inactive conformations of target proteins by ix attained by binding of ligands is huge in cGMP bound state rather than CyaB2 GAF domain ‘preferred’ cAMP bound state. In addition, bound cGMP conferred an additional conformational change on C-terminal !5 helix region of the protein. This change in specificity might be due to the isolated domain. Studies with stereospecific analogs revealed that presence of sulfur at either equatorial or axial position was not able to order the protein structure and the interaction between these analogs and protein was weaker. This was observed clearly from high HDX throughout the protein compared to cAMP/cGMP bound state. 147 Future Directions Structural information obtained by studying the deletion mutants of PKA RI!(91-244), RI!($91) and GAF-B domain revealed many insights in cAMP mediated conformational changes in isolated domains. However, native construct of these proteins will allow us to determine physiologically relevant cAMP mediated activation in vivo. 1. cAMP mediated conformational dynamics of full length RI! dimer and holoenzyme formed by RI! dimer and two C- subunit monomers. Studies on RI!(91-244) and RI!($91) have unraveled that RI!(91-244) exists as an ensemble of conformations and binding of cAMP will lead to ‘B’ conformation and binding of C-subunit will sample out ‘H’ conformation. Extension of our study with RI!($91) allowed us to understand the cooperativity between two CNB domains and role of CNB-B domain in the process of activation. Further studies on full length RI! dimer and physiologically relevant holoenzyme will elucidate the mode of dimerization. However, we can not get this information by using traditional methods like X-ray and NMR because of flexible dimerization domain and large size of the protein. HDX experiments with this physiological R-subunit and holoenzyme will help us in determining the structure of physiologically relevant holoenzyme. In addition, studies using CNB-B specific ligands on these full length proteins will allow us to determine step wise activation process and also role of dimerization in the process activation. 2. Elucidation of MSMEG_5458 activation in presence of acetyl-CoA to USP. Because of the lack of structural information of the MSMEG_5458, we have modeled CNB domain and transferase domain separately using MODELLER and mapped our HDX results on it. Lack of continuous full length crystal structure for MSMEG_5458 148 created a barrier to predict cAMP mediated action confidently. Crystallization of full length MSMEG_5458 with and with out cAMP and acetyl-CoA will help us completely in understanding the cAMP mediated activation of this unique protein. In addition, extending our experiments to MSMEG_5458 ortholog Rv0998 obtained from pathogenic strain of mycobacteria will help us identify the novel targets for drug development. 3. Structural characterization of physiologically relevant GAF domain of cyanobacteria. Our results have shown that isolated GAF-B domain of CyaB2 lost its specificity and displayed a more compact structure in cGMP bound state compared to cAMP bound state. We believe, HDX experiments with tandem array of GAF domains with and without cAMP and its comparison with isolated domains will elucidate the ligand specificity. Further, HDX experiments with tandem array GAF domains containing N-terminal region and comparison with tandem array of GAF domain without Nterminal region will allow us to understand the role of N-terminal region in activation process. Generation of domain specific mutants such as GAF-A and GAF-B domain mutants in different constructs which will fail to bind to ligand in one domain where as second domain occupied with ligand will reveal allosteric coupling between these two domains in the process of activation. In addition, having HDX experimental data of the above mentioned constructs will also help us understand role of dimerization between these tandem array GAF domains. 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Coupling of 1 or more of these subfamilies to the GPCRS will result in various cellular responses (Cabrera-Vera et al., 2003) Activation or inhibition of ACs is coupled to the binding of G"s or G"i to GPCR respectively and results in maintenance of cAMP synthesis in the cell (Cabrera-Vera et al., 2003) G"s bound activated AC catalyses the conversion of adenosine triphosphate (ATP) to cAMP The primary... to a release of G"s protein from G protein complex and activates AC by binding to it AC catalyze the generation of cAMP from ATP, PDE mediates the hydrolysis of cAMP to 5` AMP for termination of cAMP response Protein Kinase A (PKA) is the central downstream target for cAMP The kinase core of PKA is the catalytic (C) subunit, which exists in an inactive, tetrameric complex with a homodimeric regulatory. .. dynamics of the PKA R-subunit and cAMP- 24 dependent regulation of PKA A) Domain organization of PKA RI" B) Structure of the R-subunit in the C-subunit-bound conformation (H-conformation) (from the RA: C complex structure, PDB: 3FHI) C) Structure of the R-subunit (bound to cAMP, PDB: 1RGS) in the B-conformation D) Apo R-subunit toggles between cAMP-bound and C-subunit-bound states E) The width of the Phosphate... cooperatively facilitates binding of a second molecule of cAMP to CNB-A and leading to the release of the catalytic subunit The CNB-B thus acts as “gatekeeper” for modulating cAMP access to domain A (Kim et al., 2007) The CNB-A has also been found to be part of the direct interaction site with PKA C-subunit The CBD-A has been known to have a faster off-rate compared to CNBB for cAMP (Kim et al., 2005)... by levels of cAMP within the cell and it’s binding to the R-subunit of PKA(Johnson et al., 2001b) PKA belong to one of the largest gene families (kinase), accounting for 2% of the mammalian genome (Plowman et al., 1999) Phosphorylation by these kinases mediates most of cellular function, whereas abnormal phosphorylation is a cause or consequence of various diseases This makes PKA as one of the most... accompanied by stabilization of the CNB and linker domain alone This is in contrast to other cAMP binding proteins, where cyclic nucleotide- binding has been shown to involve elaborate allosteric relays Finally, this powerful convergence of results from BRET and HDXMS reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins, in the absence of high resolution... believe that the enhanced dynamics of these regions forms the basis for the positive cooperativity in the cAMP-dependent activation of PKA In summary, our results reveal the close allosteric coupling between CNB-A and CNB-B with the C-subunit providing important molecular insights into the function of CNB-B domain With our expertise on the cAMP-binding domain, we sought to extend our analysis to a prokaryotic... coordinate binding to the ribose 2'-OH, and the equatorial and axial oxygen atoms of cAMP are displayed in sticks Arrow A highlights the Arg 209- equatorial oxygen- Asp 170 allosteric relay in PKA RIa Arrow B highlights the hydrophobic switch mediated by Leu 203 and Ile 204 with a:B/C helices B) CNB domain of KATms was modeled in the SWISS MODEL automated server using structural coordinates of PKA RIa (PDB... that binding of one molecule of cAMP increases HDX in important regions within the second CNB-B domain Increased exchange was also seen at the interface between CNB-B and the C-subunit suggesting weakening of the R:C interface without dissociation Importantly, binding of the first molecule of cAMP greatly increases the conformational mobility/dynamics of two key regions coupling the two CNBs, the "C/C$:A . Defining the contours of cyclic nucleotide mediated regulatory switches from Prokaryotes to Eukaryotes Suguna Badireddy M. Sc (Biochemistry) A THESIS SUBMITTED FOR THE DEGREE OF. 'H' conformation. This reveals the unique importance of the equatorial oxygen of the cyclic phosphate in mediating conformational transitions from 'H' to 'B' forms highlighting. powerful convergence of results from BRET and HDXMS reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins, in the absence of high resolution