Insights into BAY 60-2770 activation and S-nitrosylation-dependent desensitization of soluble guanylyl cyclase via crystal structures of homologous nostoc H-NOX domain complexes

Abstract

The soluble guanylyl cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC's salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (β1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58-2667 bound structure reveal that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has about 10% higher fold maximal stimulation compared to BAY 58-2667. S-Nitrosylation of the BAY 60-2770 substituted Nostoc H-NOX domain causes subtle changes in the vicinity of the S-nitrosylated C122 residue. These shifts could impact the adjacent YxSxR motif and αF helix and as such potentially inhibit either heme incorporation or NO-activation of sGC and thus provide a structural basis for desensitization.

Figure 1

Chemical structures of (A) BAY 58-2667 and (B) BAY 60-2770 compounds

Figure 2

Crystal contact-mediated local conformation changes upon the C139A mutation in Ns H-NOX. Superposition of wt Ns H-NOX (gray color, PDB code 2o09) and C139A mutant (yellow color). For clarity, only molecule A of the C139A mutant structure is depicted (in yellow) whereas both molecules A and B of the wt NS H-NOX structure are shown (both in grey). C139A mutation confers changes in the loop region of a related monomer, ultimately leading to better crystal packing (and improved diffraction data).

Figure 3

Activation effects of bovine sGC by BAY 58-2667 and BAY 60-2770 in the absence and presence of ODQ (10 µM).

Figure 4

BAY 60-2770 interactions in Ns H-NOX. (A) Cartoon representation of BAY 60-2770 (in sticks) complexed to C139A Ns H-NOX (molecule A); H105 and C122 are shown in sticks. (B) 2F_o-F_c electron density contoured at 1σ in blue and omit F_o-F_c density contoured 2.5σ in green color for BAY 60-2770 within heme cavity. (C) Interactions of BAY 60-2770 with nearby H-NOX residues, hydrogen bonds are shown as dashed lines.

Figure 5

Comparison between BAY 58-2667 and BAY 60-2770 bound Ns H-NOX structures. BAY 58-2667 bound Ns H-NOX structure in blue color and BAY 60-2770 bound C139A Ns H-NOX structure in red color. (A) Side view of cartoon superposition of BAY 58 bound and BAY 60 bound H-NOX structures showing no structural change in αF-helix while its C-terminus loop region 110-114 is altered; side chains of loop region residues are shown in sticks and BAY 58 and BAY 60 drug molecules are shown in blue and red stick models, respectively. As a reference, the superimposed heme-bound protein structure of H-NOX is shown in grey. (B) Top view of figure 5 (A), distances between side chain positions are shown with double-head arrows. BAY 58-2667 and BAY 60-2770, H105 of αF-helix and loop residues - L110, S111, F112, P113, Q114 are shown in sticks.

Figure 6

Electron density maps showing S-nitrosylation of C122 of Ns H-NOX. 2F_o-F_c electron density map in blue contoured at 1σ and F_o-F_c electron density map in green contoured at 3σ. (A) Green density near C122 showing S-nitrosylation difference density of C122 prior to modeling residue as an S-nitrosylated cysteine (in molecule A). (B) 2F_o-F_c electron density map in blue contoured at 1σ of S-nitrosylated C122.

Figure 7

Structural effects of S-nitrosylation of C122 in Ns H-NOX. Superposition of BAY 60-2770 bound C139A Ns H-NOX structure (in red, molecule A) and S-nitrosylated BAY 60-2770-bound structure (in green, molecule A) (A) S-nitrosylation induces conformational changes at C-terminus of αF-helix and the residues near C122. C122-SNO shown in ball and stick model (red & blue). (B) Side view from C-terminus of αF-helix (C) Close view of C122 and its nearby residues. Relevant residues are labeled and shown in sticks.