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

Abstract

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.

Activation mechanism of human soluble guanylate cyclase by stimulators and activators

“…In addition to the YC-1 type stimulators, there are other sGC stimulators with distinct chemical structures 16 , which might bind sGC at different sites. As some mutations in the catalytic module can enhance the sGC activity 24 , 45 , it is possible that certain hypothetic molecules can bind inside the catalytic module to open the substrate binding pocket and to activate sGC. Because the conformation of catalytic module is allosterically coupled to the structure of the sensor module 46 – 48 , these hypothetic molecules might likely induce similar extended conformation of sGC.…”

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…The quaternary structure of human sGC was constructed using a hybrid approach that combines protein structure prediction tools with cryoEM experimental data, to give insight into the inter-domain communication upon NO binding . The communication between domains was further explored by molecular dynamics simulations and mutational studies coupled with luciferase reporter assays to identify hot spot linkages in the CC domain of human sGC that could be a part of the propagation of the activating signal through the CC domain when NO binds to the active site heme in the sensor domain . The conformational changes when GTP and cGMP are bound to the CAT domain of human sGC were studied using molecular dynamics simulations, and the results suggest that structural changes in the CAT domain lock and stabilize GTP within a closed pocket for cyclization.…”

Maturation, inactivation, and recovery mechanisms of soluble guanylyl cyclase

“…Only the ferrous heme-containing sGCαβ heterodimer can respond to NO to catalyze cGMP formation ( 14 , 18 , 29 ). NO binding to the ferrous heme of sGCβ breaks the proximal His-heme iron bond ( 14 , 18 ), initiating a series of structural changes that project through the heterodimer CC domains to the catalytic domains, resulting in an increased cGMP synthesis activity ( 17 , 20 , 30 ). If the sGC heme becomes oxidized to the ferric state, it is no longer activated by NO and is also more prone to dissociate from the sGC ( 18 , 31 , 32 , 33 , 34 , 35 , 36 ).…”