Physiological activation and deactivation of soluble guanylate cyclase

Abstract

Soluble guanylate cyclase (sGC) is responsible for transducing the gaseous signaling molecule nitric oxide (NO) into the ubiquitous secondary signaling messenger cyclic guanosine monophosphate in eukaryotic organisms. sGC is exquisitely tuned to respond to low levels of NO, allowing cells to respond to non-toxic levels of NO. In this review, the structure of sGC is discussed in the context of sGC activation and deactivation. The sequence of events in the activation pathway are described into a comprehensive model of in vivo sGC activation as elucidated both from studies with purified enzyme and those done in cells. This model is then used to discuss the deactivation of sGC, as well as the molecular mechanisms of pathophysiological deactivation.

Keywords: Allosteric activation; Heme cofactor; Nitric oxide; Soluble guanylate cyclase.

Fig. 1.. Domain and subdomain organization of sGC

A) Schematic of soluble guanylate cyclase domains. The four domains of the a (in orange) and p (in blue) subunits are listed above the schematic representation: heme nitric oxide and oxygen binding domain (H-NOX), Per-Arnt-Sim domain (PAS), coiled-coil domain (CC), and the catalytic domain (CAT). The approximate numbering of the Human α1 and β1 domains is shown. The heme cofactor, shown as a red diamond, binds to β1 H105. B) Motions of heme nitric oxide and oxygen binding domains (H−NOX) upon nitric oxide binding. Overlay of the Shewanella oneidensis H-NOX domain in the Fe²⁺ (wheat, PDB ID: 4U99) and Fe²⁺−NO (blue, PDB ID: 4U9B) states. Structures have been aligned by the proximal domains (residues 95–180). Displacement of the distal domain and rotation of the proximal histidine are noted with arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2.. Reconstructed full-length representation of sGC.

Using available crystal structures and single-particle electron microscopy data from Campbell et al. [40], a representation of the full-length structure of sGC was constructed. The subunits are colored as in Fig. 1A. Beginning with two representative EM volumes (left), the following crystal structures were fit into the densities: Human α1 and β1 sGC H-NOX domain homology models were derived from the bacterial H-NOX from Nostoc sp (PDB ID: 2O09); Human α1 and β1 PAS domain homology models were derived from eukaryotic PAS from C. reinhardtii (PDB ID: 4GJ4) and aligned using the Nostoc punctiforme PAS dimer (PDB ID: 2P04); Human α1 and β1 CC domain homology models and the human CAT structure (PDB ID: 4NI2) were aligned to the Mycobacterium intracellulare adenylate cyclase structure (PDB ID: 505l), chains C and D, and adjusted to fit within the volumes. These crystal structures have been fitted into the densities and are shown as ribbon diagrams (middle). The two extreme conformations of sGC are shown and labeled as “Extended” and “Contracted”. The space-filling model of both conformations is shown (right). All homology models were generated using thePhyre2 web portal [102].

Fig. 3.. Quaternary domain organization of sGC upon NO activation.

It is predicted that some spatial reorganization of the sGC domains occurs when NO activates the enzyme. A) Self-inhibition of sGC through contact of the β H-NOX domain to the α CAT domain, termed the contracted conformation. Upon NO binding, this contact would be released allowing a conformational change into the extended conformation which in full catalytic activity. B) When NO is not bound, the tertiary structure holds sGC in a non-active conformation while the quaternary structure is flexible (not depicted). Upon NO binding, structural rearrangements occur along the protein backbone, which results in full catalytic activity.

Fig. 4.. Nitric oxide dependent spectral transitions in sGC.

Both the formation of the 6c ferrous nitrosyl complex and cleavage of the iron–histidyl bond are dependent on NO concentration. A) The absorbance at 431 nm, which corresponds to Soret maximum the 5c ferrous-histidine complex, decreases with time as sGC interacts with the indicated NO concentrations (in μM). B) Dependence of the slow and the fast rates extracted from A) on NO concentration. Adapted from Ref. [29].

Fig. 5.. Nitric oxide sidedness on the heme.

A depiction of the molecular steps of NO binding to and dissociation from the heme in sGC. A) NO binds to the distal side of the heme with an association rate of k.₁ to form species (2). Rupture of the proximal histidine iron bond with the rate of k.₂ yields species (3). Note that k.₂ is dependent on NO concentration, as shown in Fig. 4. To reverse this process, the proximal histidine rebinds to the heme with a rate of k₋₂, followed by the dissociation of NO from the distal side. B) For NO to bind on the proximal side of the heme, the distal 5c species must form first (as in A). Another molecule of NO binds to the proximal side of the heme with a rate of k₃ to form species (4), followed by the cleavage of the distal NO−Fe bond with a rate of k4 to yield species (5). The reverse of this process requires either excess NO (to undergo the microscopic reverse), or another ligand (L) to bind to the distal side of the heme. Figure adapted from Ref. [66].

Fig. 6.. Kinetics of sGC transitions in the heme pocket.

The fate of the 5c iron-histidine species formed after laser photolysis of NO is depicted. A 6 ns 532 nm laser pulse, indicated by the green bar, dissociates NO. The proximal histidine rebinds to the heme with a time constant of τ₀, measured in a separate experiment. There are two routes through which the 5c His−Fe²⁺ species can proceed. In the route occurring at faster timescales (bottom), geminate recombination of NO occurs with a lifetime of 6.5 ns, followed by cleavage of the iron–histidyl bond with a life time of 0.66 [is. τ ₁ and τ ₂ do not depend on NO concentration. In the route occurring at longer timescales (top), NO from the solution diffuses into the heme pocket in a bimolecular process and binds to the heme with lifetimes of 50 is ≤ τ ₃ ≤ 250 is, followed by cleavage of the iron-histidyl bond with lifetimes of 10 ms ≤ τ ₄ ≤ 43 ms (NO concentration between 20 iM and 200 iM). Both τ ₃ and τ ₄ are dependent on the NO concentration, consistent with the stopped-flow spectroscopy data in Fig. 4. Figure adapted from Ref. [30].

Fig. 7.. Blocking of sGC cysteines prevents full enzyme activation.

A) Experimental data showing that MMTS-treated sGC will not fully activate with excess NO but will activate to a certain extent with NO and a stimulator. B) MMTS does not affect the basal activity level of sGC. C) Treatment of the MMTS-labeled enzyme with DTT reverses the inhibition. D) The activity of the MMTS-treated sGC is comparable to the activity of the 1-NO state. Figure reprinted from Ref. [70].

Fig. 8.. sGC activation and deactivation in vivo.

A model that accounts for all available data of physiological sGC activation and deactivation is depicted. For ease of representation, the sGC state with basal activity is depicted in the conformation in which the β H-NOX contacts the α sGC CAT domain. It is not known how the overall quaternary structure of sGC changes upon NO binding. The first molecule of NO that sGC encounters will bind to the heme of the H-NOX domain with picomolar affinity and remain bound. Thus, the vast majority of sGC in cells will be in the ~15% active state (1-NO). With an increase in the NO cellular concentration, NO will interact with the second site, which has nanomolar affinity. The physiologically relevant sGC states are boxed. Although the location of the second site has not yet been located yet, it is depicted here in the PAS domains. The modulation between these two states accounts for the downstream effects of NO in cells, as well as the rapid activation and deactivation of sGC observed both with purified enzyme, in cells, and in tissues.