Defining the contours of cyclic nucleaotide mediated regulatory switches from prokaryotes to eukaryotes

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. Extension of these studies to GAF domain containing PDEs will help us understand the different mode of dimerization between them (Cya B1 and B2 antiparallel and PDE parallel) and its relevance for regulation. 149 References: Abu-Abed, M., Das, R., Wang, L., and Melacini, G. (2007). Definition of an electrostatic relay switch critical for the cAMP-dependent activation of protein kinase A as revealed by the D170A mutant of RIalpha. Proteins 69, 112-124. Agarwal, N., Lamichhane, G., Gupta, R., Nolan, S., and Bishai, W.R. (2009). cAMP intoxication of macrophages by a M. tuberculosis adenylate cyclase. Nature 460, 98102. Ahuja, N., Kumar, P., and Bhatnagar, R. (2004). The adenylate cyclase toxins. Crit Rev Microbiol 30, 187-196. Amieux, P.S., and McKnight, G.S. (2002). The essential role of RI alpha in the maintenance of regulated PKA activity. Ann N Y Acad Sci 968, 75-95. Anand, G., Taylor, S.S., and Johnson, D.A. (2007). Cyclic-AMP and pseudosubstrate effects on type-I A-kinase regulatory and catalytic subunit binding kinetics. Biochemistry 46, 9283-9291. Anand, G.S., Hughes, C.A., Jones, J.M., Taylor, S.S., and Komives, E.A. (2002). Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase A. J Mol Biol 323, 377-386. Anand, G.S., Krishnamurthy, S., Bishnoi, T., Kornev, A., Taylor, S.S., and Johnson, D.A. (2010). Cyclic AMP- and (Rp)-cAMPS-induced conformational changes in a complex of the catalytic and regulatory (RI{alpha}) subunits of cyclic AMPdependent protein kinase. Mol Cell Proteomics 9, 2225-2237. Anantharaman, V., Koonin, E.V., and Aravind, L. (2001). Regulatory potential, phyletic distribution and evolution of ancient, intracellular small-molecule-binding domains. Journal of molecular biology 307, 1271-1292. Aravind, L., Mazumder, R., Vasudevan, S., and Koonin, E.V. (2002). Trends in protein evolution inferred from sequence and structure analysis. Current opinion in structural biology 12, 392-399. Aravind, L., and Ponting, C.P. (1997). The GAF domain: an evolutionary link between diverse phototransducing proteins. Trends Biochem Sci 22, 458-459. Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006). The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics 22, 195- 201. Ashby, C.D., and Walsh, D.A. (1972). Characterization of the interaction of a protein inhibitor with adenosine 3',5'-monophosphate-dependent protein kinases. I. Interaction with the catalytic subunit of the protein kinase. J Biol Chem 247, 66376642. 150 Ashby, C.D., and Walsh, D.A. (1973). Characterization of the interaction of a protein inhibitor with adenosine 3',5'-monophosphate-dependent protein kinases. II. Mechanism of action with the holoenzyme. J Biol Chem 248, 1255-1261. Badireddy, S., Yunfeng, G., Ritchie, M., Akamine, P., Wu, J., Kim, C.W., Taylor, S.S., Qingsong, L., Swaminathan, K., and Anand, G.S. (2011). Cyclic AMP analog blocks kinase activation by stabilizing inactive conformation: conformational selection highlights a new concept in allosteric inhibitor design. Mol Cell Proteomics 10, M110 004390. Bahar, I., Wallqvist, A., Covell, D.G., and Jernigan, R.L. (1998). Correlation between native-state hydrogen exchange and cooperative residue fluctuations from a simple model. Biochemistry 37, 1067-1075. Bai, Y., Milne, J.S., Mayne, L., and Englander, S.W. (1994). Protein stability parameters measured by hydrogen exchange. Proteins 20, 4-14. Basch, H., Krauss, M., and Stevens, W.J. (1991). Comparison of the electronicstructure of the P-O and P-S bonds. J Mol Structure- Theochem 81, 277–291. Bateman, R.H., Carruthers, R., Hoyes, J.B., Jones, C., Langridge, J.I., Millar, A., and Vissers, J.P. (2002). A novel precursor ion discovery method on a hybrid quadrupole orthogonal acceleration time-of-flight (Q-TOF) mass spectrometer for studying protein phosphorylation. J Am Soc Mass Spectrom 13, 792-803. Benoff, B., Yang, H., Lawson, C.L., Parkinson, G., Liu, J., Blatter, E., Ebright, Y.W., Berman, H.M., and Ebright, R.H. (2002). Structural basis of transcription activation: the CAP-alpha CTD-DNA complex. Science 297, 1562-1566. Berman, H.M., Ten Eyck, L.F., Goodsell, D.S., Haste, N.M., Kornev, A., and Taylor, S.S. (2005). The cAMP binding domain: an ancient signaling module. Proc Natl Acad Sci U S A 102, 45-50. Biswas, K.H., Sopory, S., and Visweswariah, S.S. (2008). The GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) is a sensor and a sink for cGMP. Biochemistry 47, 3534 -3543. Biswas, K.H., and Visweswariah, S.S. (2011). Distinct Allostery Induced in the Cyclic GMP-binding, Cyclic GMP-specific Phosphodiesterase (PDE5) by Cyclic GMP, Sildenafil, and Metal Ions. J Biol Chem 286, 8545- 8554. Boehr, D.D., Nussinov, R., and Wright, P.E. (2009). The role of dynamic conformational ensembles in biomolecular recognition. Nat Chem Biol 5, 789-796. Botsford, J.L. (1981). Cyclic nucleotides in procaryotes. Microbiol Rev 45, 620-642. Botsford, J.L., and Harman, J.G. (1992). Cyclic AMP in prokaryotes. Microbiol Rev 56, 100-122. 151 Brock, M., Fan, F., Mei, F.C., Li, S., Gessner, C., Woods, V.L., Jr., and Cheng, X. (2007). Conformational analysis of Epac activation using amide hydrogen/deuterium exchange mass spectrometry. J Biol Chem 282, 32256-32263. Bruder, S., Linder, J.U., Martinez, S.E., Zheng, N., Beavo, J.A., and Schultz, J.E. (2005). The cyanobacterial tandem GAF domains from the cyaB2 adenylyl cyclase signal via both cAMP-binding sites. Proceedings of the National Academy of Sciences of the United States of America 102, 3088-3092. Bruder, S., Schultz, A., and Schultz, J.E. (2006). Characterization of the tandem GAF domain of human phosphodiesterase using a cyanobacterial adenylyl cyclase as a reporter enzyme. The Journal of biological chemistry 281, 19969-19976. Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54, 905-921. Cabrera-Vera, T.M., Vanhauwe, J., Thomas, T.O., Medkova, M., Preininger, A., Mazzoni, M.R., and Hamm, H.E. (2003). Insights into G protein structure, function, and regulation. Endocr Rev 24, 765-781. Canaves, J.M., Leon, D.A., and Taylor, S.S. (2000). Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMPdependent protein kinase. Biochemistry 39, 15022-15031. Canaves, J.M., and Taylor, S.S. (2002). Classification and phylogenetic analysis of the cAMP-dependent protein kinase regulatory subunit family. J Mol Evol 54, 17-29. Charbonneau, H., Prusti, R.K., LeTrong, H., Sonnenburg, W.K., Mullaney, P.J., Walsh, K.A., and Beavo, J.A. (1990). Identification of a noncatalytic cGMP-binding domain conserved in both the cGMP-stimulated and photoreceptor cyclic nucleotide phosphodiesterases. Proceedings of the National Academy of Sciences of the United States of America 87, 288-292. Cheng, X., Phelps, C., and Taylor, S.S. (2001). Differential binding of cAMPdependent protein kinase regulatory subunit isoforms Ialpha and IIbeta to the catalytic subunit. J Biol Chem 276, 4102-4108. Cho-Chung, Y.S., and Nesterova, M.V. (2005). Tumor reversion: protein kinase A isozyme switching. Annals of the New York Academy of Sciences 1058, 76-86. Clarke, J., and Itzhaki, L.S. (1998). Hydrogen exchange and protein folding. Curr Opin Struct Biol 8, 112-118. Corbin, J.D., Cobb, C.E., Beebe, S.J., Granner, D.K., Koch, S.R., Gettys, T.W., Blackmore, P.F., Francis, S.H., and Wells, J.N. (1988). Mechanism and function of cAMP- and cGMP-dependent protein kinases. Adv Second Messenger Phosphoprotein Res 21, 75-86. 152 Corbin, J.D., Keely, S.L., and Park, C.R. (1975). The distribution and dissociation of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in adipose, cardiac, and other tissues. J Biol Chem 250, 218-225. Das, R., Esposito, V., Abu-Abed, M., Anand, G.S., Taylor, S.S., and Melacini, G. (2007). cAMP activation of PKA defines an ancient signaling mechanism. Proc Natl Acad Sci U S A 104, 93-98. Das, R., Mazhab-Jafari, M.T., Chowdhury, S., SilDas, S., Selvaratnam, R., and Melacini, G. (2008). Entropy-driven cAMP-dependent allosteric control of inhibitory interactions in exchange proteins directly activated by cAMP. J Biol Chem 283, 19691-19703. Das, R., and Melacini, G. (2007). A model for agonism and antagonism in an ancient and ubiquitous cAMP-binding domain. J Biol Chem 282, 581-593. Dass, B.K., Sharma, R., Shenoy, A.R., Mattoo, R., and Visweswariah, S.S. (2008). Cyclic AMP in mycobacteria: characterization and functional role of the Rv1647 ortholog in Mycobacterium smegmatis. J Bacteriol 190, 3824-3834. Deng, Y., Zhang, Z., and Smith, D.L. (1999). Comparison of continuous and pulsed labeling amide hydrogen exchange/mass spectrometry for studies of protein dynamics. J Am Soc Mass Spectrom 10, 675-684. Diller, T.C., Madhusudan, Xuong, N.H., and Taylor, S.S. (2001). Molecular basis for regulatory subunit diversity in cAMP-dependent protein kinase: crystal structure of the type II beta regulatory subunit. Structure 9, 73-82. Diller, T.C., Xuong, N.H., and Taylor, S.S. (2000). Type II beta regulatory subunit of cAMP-dependent protein kinase: purification strategies to optimize crystallization. Protein Expr Purif 20, 357-364. Doskeland, S.O., Maronde, E., and Gjertsen, B.T. (1993). The genetic subtypes of cAMP-dependent protein kinase--functionally different or redundant? Biochim Biophys Acta 1178, 249-258. Dostmann, W.R., and Taylor, S.S. (1991). Identifying the molecular switches that determine whether (Rp)-cAMPS functions as an antagonist or an agonist in the activation of cAMP-dependent protein kinase I. Biochemistry 30, 8710-8716. Engen, J.R., Wales, T.E., Hochrein, J.M., Meyn, M.A., 3rd, Banu Ozkan, S., Bahar, I., and Smithgall, T.E. (2008). Structure and dynamic regulation of Src-family kinases. Cell Mol Life Sci 65, 3058-3073. Englander, J.J., Calhoun, D.B., and Englander, S.W. (1979). Measurement and calibration of peptide group hydrogen-deuterium exchange by ultraviolet spectrophotometry. Anal Biochem 92, 517-524. 153 Englander, S.W., and Mayne, L. (1992). Protein folding studied using hydrogenexchange labeling and two-dimensional NMR. Annu Rev Biophys Biomol Struct 21, 243-265. Fimia, G.M., and Sassone-Corsi, P. (2001). Cyclic AMP signalling. J Cell Sci 114, 1971-1972. Finn, R.D., Mistry, J., Tate, J., Coggill, P., Heger, A., Pollington, J.E., Gavin, O.L., Gunasekaran, P., Ceric, G., Forslund, K., et al. (2010). The Pfam protein families database. Nucleic Acids Res 38, D211-222. Frey, P.A., and Sammons, R.D. (1985). Bond order and charge localization in nucleoside phosphorothioates. Science 228, 541-545. Gazdik, M.A., Bai, G., Wu, Y., and McDonough, K.A. (2009). Regulated intramacrophage and cAMP-induced gene expression in Mycobacterium tuberculosis – complex mycobacteria. Mol Microbiol 71, 434-448. Geromanos, S.J., Vissers, J.P., Silva, J.C., Dorschel, C.A., Li, G.Z., Gorenstein, M.V., Bateman, R.H., and Langridge, J.I. (2009). The detection, correlation, and comparison of peptide precursor and product ions from data independent LC-MS with data dependant LC-MS/MS. Proteomics 9, 1683-1695. Gibson, R.M., Ji-Buechler, Y., and Taylor, S.S. (1997). Interaction of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Electrostatic sites on the type Ialpha regulatory subunit. J Biol Chem 272, 16343-16350. Gill, G.N., and Garren, L.D. (1971). Role of the receptor in the mechanism of action of adenosine 3':5'-cyclic monophosphate. Proc Natl Acad Sci U S A 68, 786-790. Gross-Langenhoff, M., Hofbauer, K., Weber, J., Schultz, A., and Schultz, J.E. (2006). cAMP is a ligand for the tandem GAF domain of human phosphodiesterase 10 and cGMP for the tandem GAF domain of phosphodiesterase 11. The Journal of biological chemistry 281, 2841-2846. Hamm, H.E. (1998). The many faces of G protein signaling. J Biol Chem 273, 669672. Hamuro, Y., Anand, G.S., Kim, J.S., Juliano, C., Stranz, D.D., Taylor, S.S., and Woods, V.L., Jr. (2004). Mapping intersubunit interactions of the regulatory subunit (RIalpha) in the type I holoenzyme of protein kinase A by amide hydrogen/deuterium exchange mass spectrometry (DXMS). J Mol Biol 340, 1185-1196. Harper, S.M., Wienk, H., Wechselberger, R.W., Bos, J.L., Boelens, R., and Rehmann, H. (2008). Structural dynamics in the activation of Epac. J Biol Chem 283, 6501-6508. Heikaus, C.C., Stout, J.R., Sekharan, M.R., Eakin, C.M., Rajagopal, P., Brzovic, P.S., Beavo, J.A., and Klevit, R.E. (2008). Solution structure of the cGMP binding GAF domain from phosphodiesterase 5: insights into nucleotide specificity, dimerization, 154 and cGMP-dependent conformational change. The Journal of biological chemistry 283, 22749-22759. Herberg, F.W., Bell, S.M., and Taylor, S.S. (1993). Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states. Protein Eng 6, 771-777. Herberg, F.W., Taylor, S.S., and Dostmann, W.R. (1996). Active site mutations define the pathway for the cooperative activation of cAMP-dependent protein kinase. Biochemistry 35, 2934-2942. Ho, Y.S., Burden, L.M., and Hurley, J.H. (2000). Structure of the GAF domain, a ubiquitous signaling motif and a new class of cyclic GMP receptor. The EMBO journal 19, 5288-5299. Hoofnagle, A.N., Resing, K.A., and Ahn, N.G. (2003a). Protein analysis by hydrogen exchange mass spectrometry. Annu Rev Biophys Biomol Struct 32, 1-25. Hoofnagle, A.N., Resing, K.A., and Ahn, N.G. (2003b). Protein analysis by hydrogen exchange mass spectrometry. Annual review of biophysics and biomolecular structure 32, 1-25. Houde, D., Berkowitz, S.A., and Engen, J.R. (2011). The utility of hydrogen/deuterium exchange mass spectrometry in biopharmaceutical comparability studies. J Pharm Sci 100, 2071-2086. Huang, L.J., and Taylor, S.S. (1998). Dissecting cAMP binding domain A in the RIalpha subunit of cAMP-dependent protein kinase. Distinct subsites for recognition of cAMP and the catalytic subunit. J Biol Chem 273, 26739-26746. Hughes, C.A., Mandell, J.G., Anand, G.S., Stock, A.M., and Komives, E.A. (2001). Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB. J Mol Biol 307, 967-976. Hvidt, A., and Nielsen, S.O. (1966). Hydrogen exchange in proteins. Adv Protein Chem 21, 287-386. Johnson, D.A., Akamine, P., Radzio-Andzelm, E., Madhusudan, M., and Taylor, S.S. (2001a). Dynamics of cAMP-dependent protein kinase. Chemical reviews 101, 22432270. Johnson, D.A., Akamine, P., Radzio-Andzelm, E., Madhusudan, M., and Taylor, S.S. (2001b). Dynamics of cAMP-dependent protein kinase. Chem Rev 101, 2243-2270. Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47 ( Pt 2), 110-119. Kalamidas, S.A., Kuehnel, M.P., Peyron, P., Rybin, V., Rauch, S., Kotoulas, O.B., Houslay, M., Hemmings, B.A., Gutierrez, M.G., Anes, E., et al. (2006). cAMP 155 synthesis and degradation by phagosomes regulate actin assembly and fusion events: consequences for mycobacteria. J Cell Sci 119, 3686-3694. Kammer, G.M., Laxminarayana, D., and Khan, I.U. (2004). Mechanisms of deficient type I protein kinase A activity in lupus T lymphocytes. Int Rev Immunol 23, 225-244. Kannan, N., Wu, J., Anand, G.S., Yooseph, S., Neuwald, A.F., Venter, J.C., and Taylor, S.S. (2007). Evolution of allostery in the cyclic nucleotide binding module. Genome Biol 8, R264. Kaper, J.B., Nataro, J.P., and Mobley, H.L. (2004). Pathogenic Escherichia coli. Nat Rev Microbiol 2, 123-140. Kaupp, U.B., and Seifert, R. (2002). Cyclic nucleotide-gated ion channels. Physiol Rev 82, 769-824. Kim, C., Cheng, C.Y., Saldanha, S.A., and Taylor, S.S. (2007). PKA-I holoenzyme structure reveals a mechanism for cAMP-dependent activation. Cell 130, 1032-1043. Kim, C., Xuong, N.H., and Taylor, S.S. (2005). Crystal structure of a complex between the catalytic and regulatory (RIalpha) subunits of PKA. Science 307, 690696. Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R., and Thornton, J.M. (1996). AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8, 477-486. Leon, D.A., Canaves, J.M., and Taylor, S.S. (2000). Probing the multidomain structure of the type I regulatory subunit of cAMP-dependent protein kinase using mutational analysis: role and environment of endogenous tryptophans. Biochemistry 39, 5662-5671. Li, G.Z., Vissers, J.P., Silva, J.C., Golick, D., Gorenstein, M.V., and Geromanos, S.J. (2009). Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures. Proteomics 9, 1696-1719. Liang, C., and Allen, L.C. (1987). Sulfur does not form double bonds in phosphorothioate anions. J Am Chem Soc 109, 6449–6453. Linder, J.U., Bruder, S., Schultz, A., and Schultz, J.E. (2007). Changes in purine specificity in tandem GAF chimeras from cyanobacterial cyaB1 adenylate cyclase and rat phosphodiesterase 2. Febs J 274, 1514-1523. Lugnier, C. (2006). Cyclic nucleotide phosphodiesterase (PDE) superfamily: a new target for the development of specific therapeutic agents. Pharmacol Ther 109, 366398. Martinez, S.E., Bruder, S., Schultz, A., Zheng, N., Schultz, J.E., Beavo, J.A., and Linder, J.U. (2005). Crystal structure of the tandem GAF domains from a 156 cyanobacterial adenylyl cyclase: modes of ligand binding and dimerization. Proceedings of the National Academy of Sciences of the United States of America 102, 3082-3087. Martinez, S.E., Heikaus, C.C., Klevit, R.E., and Beavo, J.A. (2008). The structure of the GAF A domain from phosphodiesterase 6C reveals determinants of cGMP binding, a conserved binding surface, and a large cGMP-dependent conformational change. The Journal of biological chemistry 283, 25913-25919. Martinez, S.E., Wu, A.Y., Glavas, N.A., Tang, X.B., Turley, S., Hol, W.G., and Beavo, J.A. (2002). The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding. Proceedings of the National Academy of Sciences of the United States of America 99, 13260-13265. McAllister-Lucas, L.M., Haik, T.L., Colbran, J.L., Sonnenburg, W.K., Seger, D., Turko, I.V., Beavo, J.A., Francis, S.H., and Corbin, J.D. (1995). An essential aspartic acid at each of two allosteric cGMP-binding sites of a cGMP-specific phosphodiesterase. The Journal of biological chemistry 270, 30671-30679. McAllister-Lucas, L.M., Sonnenburg, W.K., Kadlecek, A., Seger, D., Trong, H.L., Colbran, J.L., Thomas, M.K., Walsh, K.A., Francis, S.H., Corbin, J.D., et al. (1993). The structure of a bovine lung cGMP-binding, cGMP-specific phosphodiesterase deduced from a cDNA clone. The Journal of biological chemistry 268, 22863-22873. McCue, L.A., McDonough, K.A., and Lawrence, C.E. (2000). Functional classification of cNMP-binding proteins and nucleotide cyclases with implications for novel regulatory pathways in Mycobacterium tuberculosis. Genome Res 10, 204-219. McNicholl, E.T., Das, R., SilDas, S., Taylor, S.S., and Melacini, G. Communication between tandem cAMP binding domains in the regulatory subunit of protein kinase A-Ialpha as revealed by domain-silencing mutations. J Biol Chem 285, 15523-15537. Mesleh, M.F., Hunter, J.M., Schvartsburg, A.A., Schatz, G.C., and Jarrold, M.F. (1996). Structural information from ion mobility measure- ments: Effects of the longrange potential. J PhysChem 100, 16082–16086. Nambi, S., Basu, N., and Visweswariah, S.S. (2010). cAMP-regulated protein lysine acetylases in mycobacteria. J Biol Chem 285, 24313-24323. Neuwald, A.F., Aravind, L., Spouge, J.L., and Koonin, E.V. (1999). AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res 9, 27-43. Ogreid, D., and Doskeland, S.O. (1983). Cyclic nucleotides modulate the release of [3H] adenosine cyclic 3',5'-phosphate bound to the regulatory moiety of protein kinase I by the catalytic subunit of the kinase. Biochemistry 22, 1686-1696. Osborne, B.W., Wu, J., McFarland, C.J., Nickl, C.K., Sankaran, B., Casteel, D.E., Woods, V.L., Jr., Kornev, A.P., Taylor, S.S., et al. (2011). Crystal Structure of 157 cGMP-Dependent Protein Kinase Reveals Novel Site of Interchain Communication. Structure19, 1317-1327. Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276, 307-326. Padh, H., and Venkitasubramanian, T.A. (1976). Cyclic adenosine 3', 5'monophosphate in mycobacteria. Indian J Biochem Biophys 13, 413-414. Perrakis, A., Morris, R., and Lamzin, V.S. (1999). Automated protein model building combined with iterative structure refinement. Nat Struct Biol 6, 458-463. Petersen, T.N., Lundegaard, C., Nielsen, M., Bohr, H., Bohr, J., Brunak, S., Gippert, G.P., and Lund, O. (2000). Protein secondary structure: category assignment and predictability. Proteins 41 17 -20. Pfleger, K.D., and Eidne, K.A. (2006). Illuminating insights into protein protein interactions using bioluminescence resonansce energy transfer (BRET). Nat Methods 3, 165-174. Plowman, G.D., Sudarsanam, S., Bingham, J., Whyte, D., and Hunter, T. (1999). The protein kinases of Caenorhabditis elegans: a model for signal transduction in multicellular organisms. Proceedings of the National Academy of Sciences of the United States of America 96, 13603-13610. Podobnik, M., Tyagi, R., Matange, N., Dermol, U., Gupta, A.K., Mattoo, R., Seshadri, K., and Visweswariah, S.S. (2009). A mycobacterial cyclic AMP phosphodiesterase that moonlights as a modifier of cell wall permeability. J Biol Chem 284 32846 32857. Ponting, C.P., and Aravind, L. (1997). PAS: a multifunctional domain family comes to light. Current biology : CB 7, R674-677. Popovych, N., Sun, S., Ebright, R.H., and Kalodimos, C.G. (2006). Dynamically driven protein allostery. Nat Struct Mol Biol 13, 831-838. Popovych, N., Tzeng, S.R., Tonelli, M., Ebright, R.H., and Kalodimos, C.G. (2009). Structural basis for cAMP-mediated allosteric control of the catabolite activator protein. Proc Natl Acad Sci U S A 106, 6927- 6932. Poteet-Smith, C.E., Corbin, J.D., and Francis, S.H. (1997). The pseudosubstrate sequences alone are not sufficient for potent autoinhibition of cAMP- and cGMPdependent protein kinases as determined by synthetic peptide analysis. Adv Second Messenger Phosphoprotein Res 31, 219-235. Raschke, T.M., and Marqusee, S. (1998). Hydrogen exchange studies of protein structure. Curr Opin Biotechnol 9, 80-86. 158 Rehmann, H., Prakash, B., Wolf, E., Rueppel, A., de Rooij, J., Bos, J.L., and Wittinghofer, A. (2003). Structure and regulation of the cAMP-binding domains of Epac2. Nat Struct Biol 10, 26-32. Rehmann, H., Wittinghofer, A., and Bos, J.L. (2007). Capturing cyclic nucleotides in action: snapshots from crystallographic studies. Nat Rev Mol Cell Biol 8, 63-73. Rickman, L., Scott, C., Hunt, D.M., Hutchinson, T., Menendez, M.C., Whalan, R., Hinds, J., Colston, M.J., Green, J., and Buxton, R.S. (2005 ). A member of the cAMP receptor protein family of transcription regulators in Mycobacterium tuberculosis is required for virulence in mice and controls transcription of the rpfA gene coding for a resuscitation promoting factor. Mol Microbiol 56 1274 -1286. Ringheim, G.E., Saraswat, L.D., Bubis, J., and Taylor, S.S. (1988). Deletion of cAMP-binding site B in the regulatory subunit of cAMP-dependent protein kinase alters the photoaffinity labeling of site A. J Biol Chem 263, 18247-18252. Ruotolo, B.T., Benesch, J.L., Sandercock, A.M., Hyung, S.J., and Robinson, C.V. (2008). Ion mobility-mass spectrometry analysis of large protein complexes. Nat Protoc 3, 1139-1152. Ruotolo, B.T., Giles, K., Campuzano, I., Sandercock, A.M., Bateman, R.H., and Robinson, C.V. (2005). Evidence for macromolecular protein rings in the absence of bulk water. Science 310, 1658-1661. Schultz, J.E. (2009). Structural and biochemical aspects of tandem GAF domains. Handb Exp Pharmacol 191, 93-109. Schwede, F., Christensen, A., Liauw, S., Hippe, T., Kopperud, R., Jastorff, B., and Doskeland, S.O. (2000). 8-Substituted cAMP analogues reveal marked differences in adaptability, hydrogen bonding, and charge accommodation between homologous binding sites (AI/AII and BI/BII) in cAMP kinase I and II. Biochemistry 39, 88038812. Scott, J.D. (1991). Cyclic nucleotide-dependent protein kinases. Pharmacology & therapeutics 50, 123-145. Shabb, J.B., Ng, L., and Corbin, J.D. (1990). Mutating protein kinase cAMP-binding sites into cGMP-binding sites. Mechanism of cGMP selectivity. J Biol Chem 265, 16031-16034. Shen, Z., Li, P., Ni, R.J., Ritchie, M., Yang, C.P., Liu, G.F., Ma, W., Liu, G.J., Ma, L., Li, S.J., et al. (2009). Label-free quantitative proteomics analysis of etiolated maize seedling leaves during greening. Mol Cell Proteomics 8, 2443-2460. Shenoy, A.R., Sreenath, N., Podobnik, M., Kovacevic, M., and S., V.S. (2005). The Rv0805 gene from Mycobacterium tuberculosis encodes a 3`,5`-cyclic nucleotide phosphodiesterase: biochemical and mutational analysis. . Biochemistry 44, 1569515704. 159 Shenoy, A.R., and Visweswariah, S.S. (2003). Site-directed mutagenesis using a single mutagenic oligonucleotide and DpnI digestion of template DNA. Anal Biochem 319, 335- 336. Shenoy, A.R., and Visweswariah, S.S. (2006). New messages from old messengers: cAMP and mycobacteria. Trends Microbiol 14, 543-550. Silva, J.C., Denny, R., Dorschel, C.A., Gorenstein, M., Kass, I.J., Li, G.Z., McKenna, T., Nold, M.J., Richardson, K., Young, P., et al. (2005). Quantitative proteomic analysis by accurate mass retention time pairs. Anal Chem 77, 2187-2200. Smith, R.S., Wolfgang, M.C., and Lory, S. (2004). An adenylate cyclase-controlled signaling network regulates Pseudomonas aeruginosa virulence in a mouse model of acute pneumonia. Infect Immun 72, 1677-1684. Soderling, S.H., and Beavo, J.A. (2000). Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions. Curr Opin Cell Biol 12, 174-179. Sopory, S., Balaji, S., Srinivasan, N., and Visweswariah, S.S. (2003). Modeling and mutational analysis of the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase, PDE5. FEBS Lett 539, 161-166. Stapleton, M., Haq, I., Hunt, D.M., Arnvig, K.B., Artymiuk, P.J., Buxton, R.S., and Green, J. (2010). Mycobacterium tuberculosis cAMP Receptor Protein (Rv3676) Differs from the Escherichia coli Paradigm in Its cAMP Binding and DNA Binding Properties and Transcription Activation Properties. J Biol Chem 285, 7016 7027, 7016- 7027. Su, Y., Dostmann, W.R., Herberg, F.W., Durick, K., Xuong, N.H., Ten Eyck, L., Taylor, S.S., and Varughese, K.I. (1995). Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains. Science 269, 807-813. Susstrunk, U., Pidoux, J., Taubert, S., Ullmann, A., and Thompson, C.J. (1998). Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor. Mol Microbiol 30, 33-46. Takano, K., Yasufuku-Takano, J., Morita, K., Mori, S., Takei, M., Osamura, R.Y., Teramoto, A., and Fujita, T. (2009). Evidence that PKA activity is constitutively activated in human GH-secreting adenoma cells in a patient with Carney complex harbouring a PRKAR1A mutation. Clin Endocrinol (Oxf) 70, 769-775. Taylor, S.S., Buechler, J.A., and Yonemoto, W. (1990). cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu Rev Biochem 59, 971-1005. Turko, I.V., Haik, T.L., McAllister-Lucas, L.M., Burns, F., Francis, S.H., and Corbin, J.D. (1996). Identification of key amino acids in a conserved cGMP-binding site of cGMP-binding phosphodiesterases. A putative NKXnD motif for cGMP binding. The Journal of biological chemistry 271, 22240-22244. 160 Uetrecht, C., Rose, R.J., van Duijn, E., Lorenzen, K., and Heck, A.J. (2010). Ion mobility mass spectrometry of proteins and protein assemblies. Chem Soc Rev 39, 1633-1655. Wales, T.E., and Engen, J.R. (2006). Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrom Rev 25, 158-170. Wales, T.E., Fadgen, K.E., Gerhardt, G.C., and Engen, J.R. (2008). High-speed and high-resolution UPLC separation at zero degrees Celsius. Anal Chem 80, 6815-6820. Wang, H., Robinson, H., and Ke, H. (2010). Conformation changes, N-terminal involvement, and cGMP signal relay in the phosphodiesterase-5 GAF domain. The Journal of biological chemistry 285, 38149-38156. Weber, I.T., Takio, K., Titani, K., and Steitz, T.A. (1982). The cAMP-binding domains of the regulatory subunit of cAMP-dependent protein kinase and the catabolite gene activator protein are homologous. Proc Natl Acad Sci U S A 79, 76797683. Weis, D.D., Engen, J.R., and Kass, I.J. (2006). Semi-automated data processing of hydrogen exchange mass spectra using HX-Express. J Am Soc Mass Spectrom 17, 1700-1703. Woodward, C., Simon, I., and Tuchsen, E. (1982). Hydrogen exchange and the dynamic structure of proteins. Mol Cell Biochem 48, 135-160. Xu, H., Hegde, S.S., and Blanchard, J.S. (2011). The Reversible Acetylation and Inactivation of Mycobacterium tuberculosis Acetyl-CoA Synthetase is Dependent on cAMP. Biochemistry 50, 5883- 5892. Zheng, J., Knighton, D.R., ten Eyck, L.F., Karlsson, R., Xuong, N., Taylor, S.S., and Sowadski, J.M. (1993). Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with MgATP and peptide inhibitor. Biochemistry 32, 21542161. Zoraghi, R., Corbin, J.D., and Francis, S.H. (2004). Properties and functions of GAF domains in cyclic nucleotide phosphodiesterases and other proteins. Mol Pharmacol 65, 267-278. 161 162 [...]... between levels of expression of these isoforms leads to malignancy of cells (Cho-Chung and Nesterova, 2005) The expression and association pattern of these tissue specific isoforms are thought to be responsible for various cAMP mediated responses in the cell (Taylor et al., 1990) There are two classes of physiological inhibitors of C-subunit: One is the R subunit which acts as receptor for cAMP to activate... 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