Please use this identifier to cite or link to this item: https://doi.org/10.1074/mcp.M110.004390
Title: Cyclic AMP analog blocks kinase activation by stabilizing inactive conformation: Conformational selection highlights a new concept in allosteric inhibitor design
Authors: Badireddy, S.
Yunfeng, G. 
Ritchie, M.
Akamine, P.
Wu, J.
Kim, C.W.
Taylor, S.S.
Qingsong, L. 
Swaminathan, K. 
Anand, G.S. 
Issue Date: Mar-2011
Citation: Badireddy, S.,Yunfeng, G.,Ritchie, M.,Akamine, P.,Wu, J.,Kim, C.W.,Taylor, S.S.,Qingsong, L.,Swaminathan, K.,Anand, G.S. (2011-03). Cyclic AMP analog blocks kinase activation by stabilizing inactive conformation: Conformational selection highlights a new concept in allosteric inhibitor design. Molecular and Cellular Proteomics 10 (3) : -. ScholarBank@NUS Repository. https://doi.org/10.1074/mcp.M110.004390
Abstract: The regulatory (R) subunit of protein kinase A serves to modulate the activity of protein kinase A in a cAMP-dependent manner and exists in two distinct and structurally dissimilar, end point 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((R p)-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 amide exchange 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 spanning both the agonist and antagonist-bound states. Thus contrary to earlier studies that explained the basis for cAMP action through "induced fit" alone, we report evidence for conformational selection, where the ligand-free apo form of the R-subunit exists as an ensemble of both B and H conformations. Although 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 Rp-cAMPS are those that preferentially "select" inactive conformations of target proteins by satisfying all "binding" constraints alone without inducing conformational changes necessary for activation. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
Source Title: Molecular and Cellular Proteomics
URI: http://scholarbank.nus.edu.sg/handle/10635/113125
ISSN: 15359476
DOI: 10.1074/mcp.M110.004390
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