Cyclic AMP Analog Blocks Kinase Activation by Stabilizing Inactive Conformation: Conformational Selection Highlights a New Concept in Allosteric Inhibitor Design*□S

The regulatory (R) subunit of protein kinase A serves to modulate the activity of protein kinase A in a cAMP-de-pendent 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 ex-change and ion mobility mass spectrometry, applied to the study of a stereospecific cAMP phosphorothioate an-alog and antagonist((Rp)-cAMPS). X-ray crystallography shows cAMP-bound R-subunit in the B form but surpris-ingly the antagonist Rp-cAMPS-bound R-subunit crystal-lized in the H conformation, which was previously as-sumed 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 an-tagonist-bound states. Thus contrary to earlier studies that explained the basis for cAMP action through “in-duced 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 transi-tions 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 ” inac-tive conformations of target proteins by satisfying all “binding ” constraints alone without inducing conforma-tional changes necessary for activation. Molecular &amp