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.

Signaling through dynamic linkers as revealed by PKA

“…The R construct spanning the IS, the linker, and CBD-A [i.e., RIα (91-244) or R A in short, Fig. 1A] exhibits affinities for the catalytic subunit of PKA (C) and for cAMP as well as structures that are similar to those observed for longer RIα constructs (26)(27)(28)(29). R A therefore inhibits C in a cAMP-dependent manner (27)(28)(29) and constitutes a functional regulatory unit.…”

“…1 B and D) (32). In apo R A , the H and B states coexist in a dynamic equilibrium (28), but their C vs. cAMP selectivity patterns are markedly different due to the H vs. B differences at the level of the α-subdomain. The C subunit exhibits higher affinity for the H-rather than the B state, whereas cAMP binds the B state more tightly than the H state, i.e., H and B represent the inactive and active forms of R A , respectively.…”

“…The C subunit exhibits higher affinity for the H-rather than the B state, whereas cAMP binds the B state more tightly than the H state, i.e., H and B represent the inactive and active forms of R A , respectively. As a result of the B selectivity of cAMP, cAMP binding to apo R A lowers the population of the H state, causing an effective weakening of the R:C interface and leading to the activation of PKA.Whereas the crystal structures of the H and B states of R A (28, 31, 32) have been pivotal in understanding the cAMP-dependent activation of PKA, the allosteric role of the flexible R A linker [i.e., RIα (99-118)] remains elusive due to lack of electron density and to crystal packing for several linker residues (28,32,33). Does the R A linker control the cAMP-dependent activation of PKA?…”

“…1 B-E), providing the first picture of an R:C complex and revealing that CBD-A is composed of a structurally invariant β-subdomain and a more dynamic α-subdomain (27,28,31,32). The α-subdomain tertiary structure changes dramatically depending on whether R A is bound to C or to cAMP (Fig.…”

“…Whereas the crystal structures of the H and B states of R A (28, 31, 32) have been pivotal in understanding the cAMP-dependent activation of PKA, the allosteric role of the flexible R A linker [i.e., RIα (99-118)] remains elusive due to lack of electron density and to crystal packing for several linker residues (28,32,33). Does the R A linker control the cAMP-dependent activation of PKA?…”