The pseudokinase domains of guanylyl cyclase–A and –B allosterically increase the affinity of their catalytic domains for substrate

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Sci. Signal.  29 Jan 2019:
Vol. 12, Issue 566, eaau5378
DOI: 10.1126/scisignal.aau5378

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Allosteric regulation through pseudokinase domains

Pseudokinase domains are similar to kinase domains, but they lack catalytic activity. Although many domains originally classified as pseudokinases were subsequently shown to have limited or context-dependent kinase activity, some act as scaffolds or allosteric regulators. The natriuretic peptide receptors GC-A and GC-B contain both pseudokinase and guanylyl cyclase domains. By combining homology modeling with biochemical analysis of mutant proteins, Edmund et al. found that the pseudokinase domains of GC-A and GC-B were allosteric regulators of the guanylyl cyclase domains. ATP bound to and stabilized the pseudokinase domain similarly to its stabilization of the active conformation of the prototypical kinase PKA. This, in turn, stimulated the receptors’ guanylyl cyclase activities. Thus, the pseudokinase domains of GC-A and GC-B promote the catalytic activity of these receptors through an allosteric mechanism that is conserved with catalytically active kinases.


Natriuretic peptides regulate multiple physiologic systems by activating transmembrane receptors containing intracellular guanylyl cyclase domains, such as GC-A and GC-B, also known as Npr1 and Npr2, respectively. Both enzymes contain an intracellular, phosphorylated pseudokinase domain (PKD) critical for activation of the C-terminal cGMP-synthesizing guanylyl cyclase domain. Because ATP allosterically activates GC-A and GC-B, we investigated how ATP binding to the PKD influenced guanylyl cyclase activity. Molecular modeling indicated that all the residues of the ATP-binding site of the prototypical kinase PKA, except the catalytic aspartate, are conserved in the PKDs of GC-A and GC-B. Kinase-inactivating alanine substitutions for the invariant lysine in subdomain II or the aspartate in the DYG-loop of GC-A and GC-B failed to decrease enzyme phosphate content, consistent with the PKDs lacking kinase activity. In contrast, both mutations reduced enzyme activation by blocking the ability of ATP to decrease the Michaelis constant without affecting peptide-dependent activation. The analogous lysine-to-alanine substitution in a glutamate-substituted phosphomimetic mutant form of GC-B also reduced enzyme activity, consistent with ATP stimulating guanylyl cyclase activity through an allosteric, phosphorylation-independent mechanism. Mutations designed to rigidify the conserved regulatory or catalytic spines within the PKDs increased guanylyl cyclase activity, increased sensitivity to natriuretic peptide, or reduced the Michaelis constant in the absence of ATP, consistent with ATP binding stabilizing the PKD in a conformation analogous to that of catalytically active kinases. We conclude that allosteric mechanisms evolutionarily conserved in the PKDs promote the catalytic activation of transmembrane guanylyl cyclases.

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