Instability in a coiled‐coil signaling helix is conserved for signal transduction in soluble guanylyl cyclase

Abstract

How nitric oxide (NO) activates its primary receptor, α1/β1 soluble guanylyl cyclase (sGC or GC‐1), remains unknown. Likewise, how stimulatory compounds enhance sGC activity is poorly understood, hampering development of new treatments for cardiovascular disease. NO binding to ferrous heme near the N‐terminus in sGC activates cyclase activity near the C‐terminus, yielding cGMP production and physiological response. CO binding can also stimulate sGC, but only weakly in the absence of stimulatory small‐molecule compounds, which together lead to full activation. How ligand binding enhances catalysis, however, has yet to be discovered. Here, using a truncated version of sGC from Manduca sexta, we demonstrate that the central coiled‐coil domain, the most highly conserved region of the ~150,000 Da protein, not only provides stability to the heterodimer but is also conformationally active in signal transduction. Sequence conservation in the coiled coil includes the expected heptad‐repeating pattern for coiled‐coil motifs, but also invariant positions that disfavor coiled‐coil stability. Full‐length coiled coil dampens CO affinity for heme, while shortening of the coiled coil leads to enhanced CO binding. Introducing double mutation αE447L/βE377L, predicted to replace two destabilizing glutamates with leucines, lowers CO binding affinity while increasing overall protein stability. Likewise, introduction of a disulfide bond into the coiled coil results in reduced CO affinity. Taken together, we demonstrate that the heme domain is greatly influenced by coiled‐coil conformation, suggesting communication between heme and catalytic domains is through the coiled coil. Highly conserved structural imperfections in the coiled coil provide needed flexibility for signal transduction.

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

“…Interestingly, our independent MD simulations also showed an asymmetric conformation of the flaps in mutant αβGC cat (see above). Such asymmetric arrangement may allow tight coupling of the catalytic subunits with the coiled-coil domain thought to transmit the activation signal to the active site (45–47).…”

Solution structures of the Shewanella woodyiH‐NOX protein in the presence and absence of soluble guanylyl cyclase stimulator IWP‐051

“…PCC 7120 ( Ns H‐NOX), Shewanella oneidensis ( So H‐NOX ), Shewanella woodyi ( Sw H‐NOX ), and Cb SONO . More recently, moderate resolution (3.8–5.8 Å) cryo‐electron microscopy models for sGC in inactive and active conformations show sGC undergoing a dramatic change in conformation upon activation that involves kink‐straightening and repacking of the coiled‐coil domain, 16,17 which has conserved positions of instability to facilitate activation 18 . The H‐NOX domain lies along the coiled coil domain in these structures, and density for YC‐1 can be seen at the interface of the H‐NOX and coiled‐coil domains 17 …”

cGMP: a unique 2nd messenger molecule – recent developments in cGMP research and development

“…Following photoaffinity labeling with IW-854 and subsequent mass spectrometry, the binding site for stimulator compounds was identified on the β1 subunit. Additional studies were performed to find out how the gaseous activators NO and CO enhance catalysis (Weichsel et al 2019). A stabilizing mutation within the CC domain reduces CO affinity 5-fold whereas shortened CC domains enhance CO binding.…”