Genetic alterations in the PRKACA gene coding for the catalytic α subunit of the cAMP-dependent protein kinase A (PKA-C) are linked to cortisol-secreting adrenocortical adenomas, resulting in Cushing’s syndrome. Among those, a single mutation (L205R) has been found in up to 67% of patients. Because the x-ray structures of the wild-type and mutant kinases are essentially identical, the mechanism explaining aberrant function of this mutant remains under active debate. Using NMR spectroscopy, thermodynamics, kinetic assays, and molecular dynamics simulations, we found that this single mutation causes global changes in the enzyme, disrupting the intramolecular allosteric network and eliciting losses in nucleotide/pseudo-substrate binding cooperativity. Remarkably, by rewiring its internal allosteric network, PKA-CL205R is able to bind and phosphorylate non-canonical substrates, explaining its changes in substrate specificity. Both the lack of regulation and change in substrate specificity reveal the complex role of this mutated kinase in the formation of cortisol-secreting adrenocortical adenomas.

An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera’s allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.

ATP-competitive inhibitors are currently the largest class of clinically approved drugs for protein kinases. By targeting the ATP-binding pocket, these compounds block the catalytic activity, preventing substrate phosphorylation. A problem with these drugs, however, is that inhibited kinases may still recognize and bind downstream substrates, acting as scaffolds or binding hubs for signaling partners. Here, using protein kinase A as a model system, we show that chemically different ATP-competitive inhibitors modulate the substrate binding cooperativity by tuning the conformational entropy of the kinase and shifting the populations of its conformationally excited states. Since we found that binding cooperativity and conformational entropy of the enzyme are correlated, we propose a new paradigm for the discovery of ATP-competitive inhibitors, which is based on their ability to modulate the allosteric coupling between nucleotide and substrate-binding sites.

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The sarcoplasmic reticulum (SR) Ca 2+ -ATPase (SERCA) plays a central role in muscle contractility and nonshivering thermogenesis. SERCA is regulated by sarcolipin (SLN), a single-pass membrane protein that uncouples Ca 2+ transport from ATP hydrolysis, promoting futile enzymatic cycles and heat generation. The molecular determinants for regulating heat release by the SERCA/SLN complex are unclear. Using thermocalorimetry, chemical cross-linking, and solid-state NMR spectroscopy in oriented phospholipid bicelles, we show that SERCA's functional uncoupling and heat release rate are dictated by specific SERCA/SLN intramembrane interactions, with the carboxyl-terminal residues anchoring SLN to the SR membrane in an inhibitory topology. Systematic deletion of the carboxyl terminus does not prevent the SERCA/SLN complex formation but reduces uncoupling in a graded manner. These studies emphasize the critical role of lipids in defining the active topology of SLN and modulating the heat release rate by the SERCA/SLN complex, with implications in fat metabolism and basal metabolic rate.