Synergistic mutations in soluble guanylyl cyclase (sGC) reveal a key role for interfacial regions in the sGC activation mechanism.

Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) and a central component of the NO-cGMP pathway, critical to cardiovascular function. NO binding to the N-terminal sensor domain in sGC enhances the cyclase activity of the C-terminal catalytic domain. Our understanding of the structural elements regulating this signaling cascade is limited, hindering structure-based drug design efforts that target sGC to improve the management of cardiovascular diseases. Conformational changes are thought to propagate the NO-binding signal throughout the entire sGC heterodimer, via its coiled-coil domain, to reorient the catalytic domain into an active conformation. To identify the structural elements involved in this signal transduction cascade, here we optimized a cGMP-based luciferase assay that reports on heterologous sGC activity in Escherichia coli and identified several mutations that activate sGC. These mutations resided in the dorsal flaps, dimer interface, and GTP-binding regions of the catalytic domain. Combinations of mutations from these different elements synergized, resulting in even greater activity and indicating a complex cross-talk among these regions. Molecular dynamics simulations further revealed conformational changes underlying the functional impact of these mutations. We propose that the interfacial residues play a central role in the sGC activation mechanism by coupling the coiled-coil domain to the active site via a series of hot spots. Our results provide new mechanistic insights not only into the molecular pathway for sGC activation but also for other members of the larger nucleotidyl cyclase family.