NO and CO differentially activate soluble guanylyl cyclase via a heme pivot-bend mechanism

Abstract

Diatomic ligand discrimination by soluble guanylyl cyclase (sGC) is paramount to cardiovascular homeostasis and neuronal signaling. Nitric oxide (NO) stimulates sGC activity 200-fold compared with only four-fold by carbon monoxide (CO). The molecular details of ligand discrimination and differential response to NO and CO are not well understood. These ligands are sensed by the heme domain of sGC, which belongs to the heme nitric oxide oxygen (H-NOX) domain family, also evolutionarily conserved in prokaryotes. Here we report crystal structures of the free, NO-bound, and CO-bound H-NOX domains of a cyanobacterial homolog. These structures and complementary mutational analysis in sGC reveal a molecular ruler mechanism that allows sGC to favor NO over CO while excluding oxygen, concomitant to signaling that exploits differential heme pivoting and heme bending. The heme thereby serves as a flexing wedge, allowing the N-terminal subdomain of H-NOX to shift concurrent with the transition of the six- to five-coordinated NO-bound state upon sGC activation. This transition can be modulated by mutations at sGC residues 74 and 145 and corresponding residues in the cyanobacterial H-NOX homolog.

Figure 1

Structure and domain organization of H-NOX and H-NOXA-containing proteins. (A) Schematic diagram of the H-NOX and H-NOXA domains present in proteins from animals and Nostoc cyanobacteria. The sGC subunits contain additional coiled-coil (CC) and GC domains. The purple interrupted line indicates the domain that was targeted for crystallographic studies. The PAS-like domain is termed H-NOXA, as it is often associated with H-NOX and was previously named H-NOBA (Iyer et al, 2003) before the discovery that these heme domains could also bind oxygen (Pellicena et al, 2004). The proteins transcribed adjacent to the H-NOX gene in cyanobacteria are the signal transduction histidine kinases (STHK) and the two-component hybrid sensor and regulator (2-CHSR). (B) Structure-based sequence alignment of HNOX domains. Residues in the heme cavity are labeled in orange or red, the latter if the residue is fully conserved. Additional conserved residues in the hydrophobic core are shaded yellow. Key residues for the transition from six- to five-coordinated NO bound are labeled with a ‘#' (W74 and M144). The three N-terminal helical rods are red. Residues identical to rat sGCβ1 are in bold. Conserved glycine residues are shaded green. The GenBank identifiers for the selected sequences are Nostoc sp, GI:17229770; N. punctiforme, GI:23129606; Legionella pneumophila1, GI:52841290; L. pneumophila2, GI:52629778; Vibrio Cholerae, GI:15601476; T. tengcongensis, GI:20807169; Clostridium acetobutylicum, GI:15896488; Rattus_sGCβ1, GI:28564567; Rattus_sGCβ2, GI:21956635; Caenorhabditis elegans_GCY35, GI:52782806; Drosophila melanogaster_sGCβ1, GI:7302016.

Figure 2

Crystal structure of unliganded Ns H-NOX. (A) Schematic diagram of the structure of Ns H-NOX. The three N-terminal helices αA–αC (red), the heme (blue), and H105 (green) are highlighted. The heme propionate groups attached to pyrrole rings A and D are labeled P_A and P_D, respectively. Figure generated using MOLSCRIPT and Raster3D (Kraulis, 1991; Merritt and Bacon, 1997). (B) Electronic absorption spectra of wt and mutants of Ns H-NOX. The spectra before (solid line) and after adding 200 μM SNAP (dotted line) as the NO donor are shown. The five-coordinate free heme signature peak is near 430 nm, the six-coordinated NO-bound heme signature peak is near 416 nm, and the five-coordinated NO-bound heme is near 400 nm. For the wt Ns H-NOX, a spectrum in the presence of CO was also measured (interrupted line), yielding a peak near 423 nm, indicating the presence of a six-coordinated CO-bound state. (C) Stereo figure showing electron density for the heme group in the free Ns H-NOX structure. The main chain is depicted in coil representation, with side chains in close proximity to the heme in stick representation; residues from the N-terminal residues 1–60 are shown in red. The omit ∣F_o∣−∣F_c∣ density is in blue (2.1 Å resolution, contoured at 3σ). Figure generated using BOBSCRIPT (Esnouf, 1999). (D) Superposition of the Ns and Tt H-NOX structures revealing an N-terminal domain shift for helices αA–αC. Depicted are the Ns H-NOX structure (blue), partially shifted Tt H-NOX (green; monoclinic molecule B in PDBid 1U55), and maximally shifted Tt H-NOX (red, monoclinic molecule A in PDBid 1U55) (Pellicena et al, 2004). The arrow indicates the direction of the shift.

Figure 3

Structural consequences of NO and CO binding to Ns H-NOX. (A) NO binding to the distal face of the heme in the NO-bound Ns H-NOX structure. Omit ∣F_o∣−∣F_c∣ electron density (contoured blue at 5σ) and 2∣F_o∣−∣F_c∣ electron density (contoured gray at 1 σ) for the heme and its two ligands, NO and H105, are shown. (B) As above, but for CO binding to H-NOX, omit density contoured at 4σ, 2∣F_o∣−∣F_c∣ contoured at 1σ level. (C) Molecular shifts within the heme region of Ns H-NOX upon NO and CO binding. Superpositioning of free (blue), NO-bound (yellow), CO-bound Ns H-NOX (magenta) structures reveals a shift in the heme αA, including residues 1–3, and loop αB–αC containing residues E41. Hydrogen bonds between the heme propionate and main chain of Y2 and between the main chains of G3 and E41 are shown (dashed lines). (D) CO binding to Ns H-NOX leads to increased flexibility in the loop following helix αF and C-terminal part of this helix. Depicted is a close-up view of the heme region of the free Ns-HNOX structure (left) and CO-bound Ns H-NOX structure (right). The temperature factor of the main chain is color ramped from blue (∼35 Å2), green (∼45 Å2), to orange (∼80 Å2). The heme and the H105 and F112 side chains are color coded by atom type. Figure generated using PYMOL (http://www.pymol.org).

Figure 4

Evidence for heme pivoting and heme bending in the activation mechanism of H-NOX. (A) Top (distal) view of the heme groups of the superimposed H-NOX structures. Ns H-NOX residues M144 and W74 are shown, as well as the residues interacting with the propionate groups of the heme (M1, Y2, Y134, S136, and R138) and residues belonging to αA–αC (red). The five heme moieties are color coded as in Figures 2D and 3C. The pivot directions of Fe and heme are indicated by arrows. (B) Side-view stereo figure of the heme groups of the superimposed HNOX structures. The five heme moieties are color coded as in (A). The van der Waals interactions between the A8 side chain and CZ3 atom of W74 as well as between CE atom of M144 and the propionate P_D group in the unliganded Ns H-NOX are depicted as transparent dashed lines. The position of the αA helix in Tt H-NOX molecule A and its M1 side chain are shown in transparent blue to indicate their potential clash with the W74 side chain and the propionate P_A group, respectively. (C) Western blot analysis of wt and mutant sGC proteins. Cytosols of the wt and H-NOX β1 heme domain mutants (F74W and I145M) for both the α1 and β1 subunits were probed with anti-sGC α1 and anti-sGC β1 antibodies.

Figure 5

Schematic diagram of NO- and CO-dependent activation of sGC. NO and CO bind to the distal face of the heme and cause the heme to pivot, with varying degrees, away from F74 (W74 in Ns H-NOX). The larger pivot for CO is likely a result of the need to accommodate its carbon atom, having a larger radius compared with nitrogen in NO, adjacent to F74 and CO's preference to bind perpendicular to the heme. The second step in the NO activation mechanism involves heme bending and N-subdomain movement (only helix αA is shown for illustrative purposes). Whether a similar heme bending event occurs in CO/YC-1-activated sGC is unknown and speculative. Also it is unknown whether a second NO molecule binds to the proximal side of the heme, thereby displacing the histidine ligand as well as the distal NO, as was postulated previously (Lawson et al, 2003; Russwurm and Koesling, 2004b). NO could therefore be bound to the distal or proximal face of the heme in the NO-bound five-coordinated activated state (both possibilities are depicted).