Somatic mutations in the PRKACA gene encoding the catalytic α subunit of protein kinase A (PKA-C) are responsible for cortisol-producing adrenocortical adenomas. These benign neoplasms contribute to the development of Cushing's syndrome. The majority of these mutations occur at the interface between the two lobes of PKA-C and interfere with the enzyme's ability to recognize substrates and regulatory (R) subunits, leading to aberrant phosphorylation patterns and activation. Rarely, patients with similar phenotypes carry an allosteric mutation, E31V, located at the C-terminal end of the αA-helix and adjacent to the αC-helix, but structurally distinct from the PKA-C/R subunit interface mutations. Using a combination of solution NMR, thermodynamics, kinetic assays, and molecular dynamics simulations, we show that the E31V allosteric mutation disrupts central communication nodes between the N- and C- lobes of the enzyme as well as nucleotide-substrate binding cooperativity, a hallmark for kinases' substrate fidelity and regulation. For both orthosteric (L205R and W196R) and allosteric (E31V) Cushing's syndrome mutants, the loss of binding cooperativity is proportional to the density of the intramolecular allosteric network. This structure–activity relationship suggests a possible common mechanism for Cushing's syndrome driving mutations in which decreased nucleotide/substrate binding cooperativity is linked to loss in substrate fidelity and dysfunctional regulation.
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health, GM100310 (G.V.), S10 OD021536 (G.V.) and GM046736 (J.G), the American Heart Association, 20PRE35120253 (C.W), and the National Natural Science Foundation of China No. 22007069 (Y.W.). NMR experiments were carried out at the Minnesota NMR Center and MD calculations at the Minnesota Supercomputing Institute.
© 2021 Elsevier Ltd
- Cushing's syndrome
- binding cooperativity
- cAMP-dependent protein kinase A
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't