Molecular Aspects of Soluble Guanylate Cyclase Activation and Stimulator Function

Soluble guanylate cyclases (sGCs) are heme-containing, gas-sensing proteins which catalyze the formation of cGMP from GTP. In humans, sGCs are highly selective sensors of nitric oxide (NO) and play a critical role in NO-based regulation of cardiovascular and pulmonary function. The physiological importance of sGC signaling has led to the development of drugs, known as stimulators and activators, which increase sGC catalytic function. Here we characterize a newly developed stimulator, CYR715, which is a particularly potent stimulator of <i>Manduca sexta</i> (<i>Ms</i>) sGC catalytic function even in the absence of NO, increasing activity of the NO-free enzyme to 45% of full catalytic activity. CYR715 also increased the catalytic activity of <i>Ms</i> sGC βC122A and βC122S variants, with a marked stimulation of the NO-free βC122S variant to 74% of maximum. High-resolution cryo-electron microscopy structures were solved for CYR715 bound to <i>Ms</i> sGC βC122S revealing that CYR715 occupies the same binding site as the characterized sGC stimulators YC-1 and riociguat. Additionally, the core scaffold of CYR715 makes a binding interaction with βC78 while the flexible tail can interact with αR429 or βY7 and E361. Conformational extension of sGC following NO, YC-1, or CYR715 binding was characterized using small-angle X-ray scattering, revealing that while ligand binding results in sGC extension this extension does not directly correlate to observed activity. This suggests that not all conformational extensions of sGC result in increased catalytic activity, and that effective stimulators assist in converting extension into catalytic function.