Discovery of stimulator binding to a conserved pocket in the heme domain of soluble guanylyl cyclase

Abstract

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly soughtafter therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the β1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L23W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting the bulkier tryptophan residues directly block stimulator binding.

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

“…We developed C‐terminal truncated versions of Ms sGC that lack both catalytic domains but retain the heme domain and respond to stimulator compounds, collectively referred to as Ms sGC‐NT proteins. We produce Ms sGC‐NT proteins in large quantity with fully loaded heme, allowing us to develop a model for domain arrangement, demonstrate linked‐equilibria binding between stimulator compounds and NO/CO, and localize stimulator compound binding to a site involving the β1 H‐NOX domain …”

Structure/function of the soluble guanylyl cyclase catalytic domain

“…Molecular modeling suggested binding to the supposed pseudo-symmetric site. However, earlier studies by the same group proposed binding of YC-1 to the catalytic domain [84], in contrast to the recent studies showing that these stimulators bind to the βHNOX domain [12]. Therefore, the exact binding site of these inhibitors remains elusive.…”

“…The remarkable opposite effects of the αCys595Ser (see section 4.1) and αCys595Tyr mutations on basal activity suggested that this residue is important for catalysis, despite not being part of the catalytic site. It was originally proposed that this residue is involved in YC-1 binding, but recent studies have shown that stimulators bind to the βHNOX sensor domain instead [12]. Instead, we proposed that αCys595 is part of a network of interfacial hydrogen bonds that promote an optimal orientation of both subunits for high catalytic activity [43].…”

“…Finally, other studies have also proposed that the αCys595 residue could participate in YC-1 stimulator [59] or ATP binding in the pseudo-symmetry site of the catalytic domain [66,67]. However, recent studies have unambiguously showed that stimulators bind to the βHNOX domain of GC-1 [12]. …”

“…Ongoing clinical trials are using ataciguat as a treatment for aortic stenosis due to calcification. More recently, studies using the biotinylated IWP-854 GC-1 stimulator helped to identify a conserved binding site for other GC-1 stimulators in the βHNOX NO-sensor domain [12]. Elucidating this binding region could provide the foundation for a class of novel GC-1 stimulators.…”