title: Cyclic AMP analog blocks kinase activation by stabilizing inactive conformation: conformational selection highlights a new concept in allosteric inhibitor design.
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((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 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.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore 117543.
authors.forename: Suguna
authors.initials: S
authors.lastname: Badireddy
authors.forename: Gao
authors.initials: G
authors.lastname: Yunfeng
authors.forename: Mark
authors.initials: M
authors.lastname: Ritchie
authors.forename: Pearl
authors.initials: P
authors.lastname: Akamine
authors.forename: Jian
authors.initials: J
authors.lastname: Wu
authors.forename: Choel W
authors.initials: CW
authors.lastname: Kim
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
authors.forename: Lin
authors.initials: L
authors.lastname: Qingsong
authors.forename: Kunchithapadam
authors.initials: K
authors.lastname: Swaminathan
authors.forename: Ganesh S
authors.initials: GS
authors.lastname: Anand
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D015195:Drug Design; D004795:Enzyme Stability / Q000187:drug effects; D006860:Hydrogen Bonding / Q000187:drug effects; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011487:Protein Conformation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity
chemical_list: D001051:Apoenzymes; D047428:Protein Kinase Inhibitors; D021122:Protein Subunits; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases