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.

利钠肽通过激活含有细胞内鸟苷酸环化酶结构域的跨膜受体来调节多个生理系统，例如 GC-A 和 GC-B，也分别称为 Npr1 和 Npr2。这两种酶都含有一个细胞内的磷酸化假激酶结构域 (PKD)，这对于激活 C 端 cGMP 合成鸟苷酸环化酶结构域至关重要。因为 ATP 变构激活 GC-A 和 GC-B，我们研究了 ATP 与 PKD 的结合如何影响鸟苷酸环化酶活性。分子模型表明，原型激酶 PKA 的 ATP 结合位点的所有残基，除了催化天冬氨酸，在 GC-A 和 GC-B 的 PKD 中都是保守的。亚结构域 II 中不变赖氨酸或 GC-A 和 GC-B DYG 环中天冬氨酸的激酶失活丙氨酸取代未能降低酶磷酸盐含量，这与缺乏激酶活性的 PKD 一致。相反，两种突变都通过阻断 ATP 降低米氏常数而不影响肽依赖性活化的能力来降低酶活化。在 GC-B 的谷氨酸取代的拟磷突变体形式中类似的赖氨酸到丙氨酸取代也降低了酶活性，这与通过变构、磷酸化非依赖性机制刺激 ATP 刺激鸟苷酸环化酶活性一致。旨在使 PKD 中保守的调节或催化刺变硬的突变增加了鸟苷酸环化酶活性，增加了对利钠肽的敏感性，或在没有 ATP 的情况下降低米氏常数，这与 ATP 结合使 PKD 稳定在类似于催化活性激酶的构象中一致。我们得出结论，PKDs 中进化上保守的变构机制促进了跨膜鸟苷酸环化酶的催化活化。