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 reveals 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.

Nitric oxide and heat shock protein 90 activate soluble guanylate cyclase by driving rapid change in its subunit interactions and heme content

“…matic activity. Although it is remarkable that BAY 60-2770 binding can mimic the effects of heme incorporation plus heme-NO binding in sGC-␤1, this behavior is completely consistent with BAY 60-2770 binding in the heme site in Nostoc H-NOX to adopt a porphyrin-like structure (21).…”

“…Although these structural changes help to show how sGC-␤1 may achieve a catalytically-active state in response to NO binding to the sGC-␤1 heme, these particular structural changes are unlikely to be the ones that weaken the apo-sGC-␤1 interaction with hsp90 because we know that heme insertion into aposGC-␤1 alone, without any NO or sGC activation, is sufficient to weaken its hsp90 association (14). However, the same subset of structural changes (21,26) is possibly associated with the process that led to a M r redistribution of sGC-␤1 in the cells. Thus, BAY 60-2770 may promote extensive protein conformational changes within apo-sGC-␤1 that lead to hsp90 dissociation, sGC-␤1 redistribution in cells, and activation of sGC enzy- FIGURE 8.…”

“…It has NO-stimulating heme insertion into the subpopulation of apo-sGC-␤1, leading to dissociation of hsp90 and to association of sGC-␣1 to form the active heterodimeric enzyme. This model is consistent with the following: (i) NO boosting sGC activation by the heme-dependent activator BAY 41-2272 and causing a concurrent loss in sGC activation by the heme-independent activator BAY-60-2770, as should occur when heme incorporates into apo-sGC-␤1; (ii) NO being unable to diminish the hsp90 association if the cells are heme-deficient and thus lacking available heme for insertion, or if the sGC-␤1 contains a mutation that impairs its ability to incorporate heme, because heme incorporation causes hsp90 dissociation (14); (iii) NO having no effect if the cell hsp90 ATPase activity is inhibited because the ATPase activity of hsp90 is needed for heme insertion to occur (14), (iv) BAY 60-2770 triggering hsp90 dissociation on its own because the drug is thought to bind in sGC-␤1 as a structural cognate of the heme⅐NO complex itself (21). Thus, hsp90 dissociation likely indicates that NO-driven heme insertion (or insertion of BAY 60-2770 in place of heme) has occurred in the apo-sGC-␤1 subunit.…”

“…Structural Insights-The crystal structures of the Nostoc H-NOX domain are regarded to be good models of the mammalian sGC-␤1 regulatory domain structure (21,26), whose structure remains to be solved. In comparing the structures of a H-NOX domain containing bound BAY 58-2667 or BAY 60-2770 with that of the drug free, heme-containing form, the authors (21, 26) identified some specific structural changes that occur with drug binding, that mainly involve the ␣-F helix and flanking residues that are located proximal to the bound heme.…”

“…This molecule binds in the heme pocket of the sGC-␤1 homolog Nostoc H-NOX domain (21) and is thought to activate sGC by triggering protein conformational changes in the sGC-␤1 subunit that mimic those caused by NO binding to the sGC-␤1 heme (21). When BAY 60-2770 was given to cells that transiently expressed the heme-free mutant sGC-␤1 H105F , or to heme-deficient RFL-6 cells that expressed endogenous apo-sGC-␤1, it caused rapid dissociation of the hsp90⅐apo-sGC-␤1 complex in both cases (Fig.…”