Solution structures of the Shewanella woodyi H-NOX protein in the presence and absence of soluble guanylyl cyclase stimulator IWP-051

Abstract

Heme-nitric oxide/oxygen binding (H-NOX) domains bind gaseous ligands for signal transduction in organisms spanning prokaryotic and eukaryotic kingdoms. In the bioluminescent marine bacterium Shewanella woodyi (Sw), H-NOX proteins regulate quorum sensing and biofilm formation. In higher animals, soluble guanylyl cyclase (sGC) binds nitric oxide with an H-NOX domain to induce cyclase activity and regulate vascular tone, wound healing and memory formation. sGC also binds stimulator compounds targeting cardiovascular disease. The molecular details of stimulator binding to sGC remain obscure but involve a binding pocket near an interface between H-NOX and coiled-coil domains. Here, we report the full NMR structure for CO-ligated Sw H-NOX in the presence and absence of stimulator compound IWP-051, and its backbone dynamics. Nonplanar heme geometry was retained using a semi-empirical quantum potential energy approach. Although IWP-051 binding is weak, a single binding conformation was found at the interface of the two H-NOX subdomains, near but not overlapping with sites identified in sGC. Binding leads to rotation of the subdomains and closure of the binding pocket. Backbone dynamics are similar across both domains except for two helix-connecting loops, which display increased dynamics that are further enhanced by compound binding. Structure-based sequence analyses indicate high sequence diversity in the binding pocket, but the pocket itself appears conserved among H-NOX proteins. The largest dynamical loop lies at the interface between Sw H-NOX and its binding partner as well as in the interface with the coiled coil in sGC, suggesting a critical role for the loop in signal transduction.

Keywords: H-NOX; NMR; drug binding; guanylate cyclase; nitric oxide; solution structure.

FIGURE 1

Chemical structures of sGC stimulators. The positions of ¹³C enrichment in IWP‐051 are indicated with stars, and overall numbering is indicated. The 5‐membered pyrazole ring is shown in the center (atoms 7, 14, 15, 22, and 24) and isoxazole ring in the upper left of the figure (atoms 5, 6, 13, 23, 26)

FIGURE 2

Backbone ensemble structures of Sw H‐NOX. (a) Ligand‐free protein. (b) Complex with IWP‐051. Secondary structure elements are labeled and heme and IWP‐051 indicated with stick representation. The structures are shown in stereo (wall‐eyed view). All structure figures were prepared with PyMOL (Schrodinger, LLC)

FIGURE 3

Binding interface between Sw H‐NOX and IWP‐051. (a) Surface and cartoon representation for the binding interface. Residues in the binding pocket are shown in blue. IWP‐051 is colored orange and shown in stick representation. (b) Close‐up view of IWP‐051 binding interactions, stick representation, with IWP‐051 shown in orange and binding pocket residues shown in blue. The single structure closest to the average of the ensemble structures is presented

FIGURE 4

Structure comparison between unliganded and IWP‐051‐bound Sw H‐NOX. (a) Unliganded and IWP‐051‐bound ensemble structures after superposing using only the C‐terminal heme subdomain (residues 63–182). Unliganded Sw H‐NOX is colored orange and the IWP‐051‐bound structure is colored purple. Shifting of the N‐terminal subdomain on binding IWP‐051 is indicated by a dashed arrow. (b) Close‐up view at the IWP‐051 binding site from the aligned structures (ensemble view upper left; ribbon cartoon of structures near the average structures, with labels, lower right). (c) Close‐up view of the heme pocket (ensemble view upper left; ribbon cartoon, lower right)

FIGURE 5

The IWP‐051 binding pocket is conserved in H‐NOX proteins. (a) Surface representation of the binding pockets from Ns H‐NOX, So H‐NOX and Ka H‐NOX. The relative position of IWP‐051 is shown by superposing the distal subdomain (residue 1–62) of Sw H‐NOX to each structure. Residues involved in the surface pocket are shown in blue stick representation and IWP‐051 in orange stick representation. (b) Multiple sequence alignment of H‐NOX domains, showing binding site residues. So: Shewanella oneidensis, Ns: Nostoc sp, Cs: Caldanaerobacter subterraneus, Hs sGC: human sGC β H‐NOX, Ka: Kordia algicida, Cb: Clostridium botulinum, Ms sGC: Manduca sexta β H‐NOX, Sw: Shewanella woodyi. Triangles indicate the residues forming the surface pockets calculated by CASTp, with red for Sw H‐NOX, blue for Ns H‐NOX, green for So H‐HNOX, and purple for Ka H‐NOX. Secondary structure is labeled on top with α helices shown as cylinders. The alignment was performed with the program T‐Coffee. ⁴³ The graphical presentation was done by the program Jalview ⁴⁴ using the Clustal X color scheme. ⁴⁵ The full alignment is shown in Figure S4

FIGURE 6

¹⁵N relaxation dynamics for native and IWP‐051‐bound Sw H‐NOX. (a) ¹⁵N longitudinal relaxation rates (R ₁) plotted against residue number. (b) ¹⁵N transverse relaxation rates (R ₂) plotted against residue number. (c) ¹⁵N[¹H] heteronuclear NOE intensities plotted against residue number. Secondary structure elements are indicated by cylinders for α helices and arrows for β strands. Data points for unliganded and IWP‐051‐bound proteins are shown as blue and orange dots, respectively. Error bars are the standard deviation from data fitting (R ₁ and R ₂ plots). Flexible loops are highlighted with purple boxes and the IWP‐051 binding site is highlighted with blue boxes

FIGURE 7

Comparison of stimulator binding sites. Shown is the cryo‐EM model for activated Manduca sGC (PDB 6PAT) ¹⁷ as a ribbon drawing with α chain in green, β chain in cyan and heme in stick format. Residues contacting Sw H‐NOX are indicated in marine blue (L12, D15, E16, L69, K72, and K73). Residues identified through photoaffinity labeling of Ms sGC to be near IWP‐051 are shown in orange (β H‐NOX domain residues 6–9 and coiled‐coil domain residues 361–362 and 365–366). ¹³ Residues within 5 Å of the density attributed to YC‐1 in the cryo‐EM model are shown in magenta (β H‐NOX domain residues V39, F77, C78 and Y112, coiled‐coil domain residues α 422–427 and β Q439). The loop displaying enhanced dynamics on IWP‐051 binding to Sw H‐NOX, and contacting the coiled coil only in the active sGC conformation, is indicated with an arrow