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

Jessica A Wales,Cheng-Yu Chen,Linda Breci,Andrzej Weichsel,Sylvie G Bernier,James E Sheppeck,Robert Solinga,Takashi Nakai,Paul A Renhowe,Joon Jung,William R Montfort

doi:10.1074/jbc.ra117.000457

Jessica A Wales, Cheng-Yu Chen + Show 9 more

Open Access

https://doi.org/10.1074/jbc.ra117.000457

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Abstract

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after 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. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that 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. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.

Highlights

We examined binding by compound PF-04447943, a phosphodiesterase 9A (PDE9A) inhibitor [28] that does not stimulate sGC
IWP-051 binding to Sw H-NOX is weaker than to Ms sGC-NT, with Kd ϭ 1.9 mM estimated for the CO complex by chemical shift perturbation NMR titration (Fig. 5B and Table S6) compared with Kd values of 0.03–3.8 ␮M for various stimulator compounds binding to the CO complexes of Ms sGC-NT constructs [20]
Most of the residues implicated in compound binding by photoaffinity labeling and NMR lie at the interface of the H-NOX subdomains (Fig. 5A), making this interface an intriguing possibility for stimulator binding

Summary

Introduction

C Range of residues that were photoaffinity-labeled in at least two sGC and/or H-NOX constructs. Many labeled peptides identified in the bacterial H-NOX proteins overlapped with those from sGC. Only labeled residues that were identified in multiple bacterial H-NOX proteins were considered to be part of the binding site.

Results

Conclusion