Mapping of the sGC Stimulator BAY 41-2272 Binding Site on H-NOX Domain and Its Regulation by the Redox State of the Heme

Abstract

Soluble guanylate cyclase (sGC) is the main receptor of nitric oxide (NO) and by converting GTP to cGMP regulates numerous biological processes. The β1 subunit of the most abundant, α1β1 heterodimer, harbors an N-terminal domain called H-NOX, responsible for heme and NO binding and thus sGC activation. Dysfunction of the NO/sGC/cGMP axis is causally associated with pathological states such as heart failure and pulmonary hypertension. Enhancement of sGC enzymatic function can be effected by a class of drugs called sGC “stimulators,” which depend on reduced heme and synergize with low NO concentrations. Until recently, our knowledge about the binding mode of stimulators relied on low resolution cryo-EM structures of human sGC in complex with known stimulators, while information about the mode of synergy with NO is still limited. Herein, we couple NMR spectroscopy using the H-NOX domain of the Nostoc sp. cyanobacterium with cGMP determinations in aortic smooth muscle cells (A7r5) to study the impact of the redox state of the heme on the binding of the sGC stimulator BAY 41-2272 to the Ns H-NOX domain and on the catalytic function of the sGC. BAY 41-2272 binds on the surface of H-NOX with low affinity and this binding is enhanced by low NO concentrations. Subsequent titration of the heme oxidant ODQ, fails to modify the conformation of H-NOX or elicit loss of the heme, despite its oxidation. Treatment of A7r5 cells with ODQ following the addition of BAY 41-2272 and an NO donor can still inhibit cGMP synthesis. Overall, we describe an analysis in real time of the interaction of the sGC stimulator, BAY 41-2272, with the Ns H-NOX, map the amino acids that mediate this interaction and provide evidence to explain the characteristic synergy of BAY 41-2272 with NO. We also propose that ODQ can still oxidize the heme in the H-NOX/NO complex and inhibit sGC activity, even though the heme remains associated with H-NOX. These data provide a more-in-depth understanding of the molecular mode of action of sGC stimulators and can lead to an optimized design and development of novel sGC agonists.

Backbone and side chain NMR assignment of the heme-nitric oxide/oxygen binding (H-NOX) domain from Nostoc punctiforme

“…2). These data, along with the other reports on bacterial H-NOX, strongly suggest that the Npun H-NOX protein domain is in the Fe(II) -H-NOX diamagnetic complex form (Boon et al, 2006;Tsai et al, 2012;Dai et al, 2012;Alexandropoulos et al, 2016;Makrynitsa et al, 2022).…”

“…The available structural studies by X-ray crystallography do not indicate significant changes between the various complexes of bacterial H-NOX domains, either in complexes with or without diatomic gases (practically all available models show significant similarity with RMSD < 0.3Å (Makrynitsa et al, 2019 ). Thus, NMR conformational and dynamic studies of other bacterial H-NOX domains are gaining interest indicating sites of interaction and other conformational modifications shedding light to analogous mechanisms of action in the human H-NOX domain (Makrynitsa et al 2022 , 2021 ; Argyriou et al, 2021 ; Erbil et al, 2009 ). Lately, structural studies have been reported regarding the NMR conformational data of Nostoc sp .…”

On a revised pharmacology of apo-soluble guanylate cyclase and its therapeutic implications

“…From a pharmacological perspective still many features of the sGCs binding site remain open: it is less well defined than that of sGCa (6,43); the molecular mechanism of its redox sensitivity amongst the many cysteines that have been mutated and analysed in sGC (6,39,(44)(45)(46)(47)(48)(49)(50) without leading to a clear pecking order with respect to redox regulation; why ODQ reduces the basal activity of sGC (21); and why the basal activity of purified apo-sGC is higher than that of sGC (51). The latter may be explained by our pilot experiment (Fig.…”

NMR Analysis Suggests Synergy between the RRM2 and the Carboxy-Terminal Segment of Human La Protein in the Recognition and Interaction with HCV IRES

“…The HCV-IRES stock concentration for the titration experiments was 5 mM, while the polyU10 stock concentration for the titration experiments was 2.5 mM. Chemical shift perturbation values were calculated using the following equation [39][40][41][42]:…”