Allostery in Recombinant Soluble Guanylyl Cyclase from Manduca sexta

Abstract

Soluble guanylyl/guanylate cyclase (sGC), the primary biological receptor for nitric oxide, is required for proper development and health in all animals. We have expressed heterodimeric fulllength and N-terminal fragments of Manduca sexta sGC in Escherichia coli, the first time this has been accomplished for any sGC, and have performed the first functional analyses of an insect sGC. Manduca sGC behaves much like its mammalian counterparts, displaying a 170-fold stimulation by NO and sensitivity to compound YC-1. YC-1 reduces the NO and CO offrates for the ϳ100-kDa N-terminal heterodimeric fragment and increases the CO affinity by ϳ50-fold to 1.7 M. Binding of NO leads to a transient six- Nitric oxide (NO)2 regulates numerous vital functions in animal physiology, including blood pressure, memory formation, platelet aggregation, and tissue development (1). The primary NO receptor is soluble guanylyl/guanylate cyclase (sGC), a heterodimeric protein of ϳ150 kDa that binds NO through a ferrous heme. NO binding stimulates cyclase activity, the production of cGMP from substrate GTP, and the subsequent amplification of NO-dependent signaling cascades (2-5). Although NO is the best described allosteric regulator of sGC, numerous other forms of regulation may also be of importance, including phosphorylation (6), nucleotide binding (7-10), calcium binding (11), nitrosylation (12, 13), and protein-protein interactions (14 -17).The primary form of sGC is an ␣1/␤1 heterodimer composed of two evolutionarily related subunits that display several recognizable domains (Fig. 1A) (18). Heme is bound to the protein through proximal ␤1 His-105. NO binding to the heme leads to proximal histidine release and stimulation of cyclase activity, presumably through a change in protein conformation (reviewed in Ref. 19). The heme-binding domain has evolved from a widespread family of bacterial proteins called H-NOX (Heme-Nitric oxide/OXygen) domain proteins, of which the structures of three are known (20 -22). The central portion of sGC contains two PAS domains (18,23), and the C-terminal region contains a catalytic domain that is very similar to that of adenylyl cyclase (24, 25).In the 1990s, the anti-platelet activity of YC-1, a benzylindazole derivative (Fig. 1B), was found to derive from its ability to bind to and stimulate sGC (26, 27), leading to a search for related compounds that might serve as sGC-targeted drugs for human health (28). The YC-1 mechanism of action remains unclear, as does the location of its binding site in the sGC protein. The nucleotide-like structure of YC-1, in conjunction with mutagenesis studies, has led to the suggestion that YC-1 binds to the cyclase domain (29), whereas cross-linking studies with YC-1-related compounds have indicated that they bind to the N-terminal domain of the ␣1 subunit (30).Studies of insect sGC have lagged behind studies of the mammalian enzyme, but, as in mammals, insect sGC plays an important physiological role. In the Manduca sexta larva (tobacco hornworm), sGC is implicated in ante...

Discovery of stimulator binding to a conserved pocket in the heme domain of soluble guanylyl cyclase

“…Ms sGC-NT constructs retain stimulator binding and response despite lacking both cyclase domains, suggesting the primary stimulator binding site resides in the N-terminal two-thirds of the protein (18)(19)(20). We examined the possibility of a secondary stimulator binding site in the catalytic domains using photoaffinity labeling of full length Hs sGC.…”

“…We previously developed truncated versions of sGC from Manduca sexta for analyses of compound-enhanced CO binding (17)(18)(19)(20), which we refer to as Ms sGC-NT (Fig. 1A).…”

“…His 6 -49-450, β1 1-380) and Ms sGC-NT21 (α1 His 6 -272-450, β1 1-380, Fig. 1A) were previously described (18)(19)(20). Plasmid pETDuet-1-NT25, coding for Ms sGC-NT25 (α1 His 6 -49-459-Strep, β1 1-389), was generated by amplifying cDNA coding for a 1 49-459 by PCR using primers F1 and R1 (primer sequences are in supplemental Table S1).…”

“…5B), but not with PDE9A inhibitor. The largest shifts cluster together near the Cterminal end of helix aA (Sw residues [14][15][16][17][18] and the N-terminal end of helix aD (Sw residues 61-76, Fig. 5C).…”

Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase

“…7 Each sGC subunit consists of four domains, an N-terminal Heme-Nitric Oxide Oxygen (H-NOX) domain 8 (also called a SONO domain), 9 a central Per-ARNT-Sim (PAS) domain, 10 a coiled-coil domain and a C-terminal catalytic cyclase domain. 11 NO binding to the heme in the [17][18][19][20] Manduca sexta sGC (Ms sGC) is highly homologous to its mammalian counterparts and responds well to YC-1, the parent compound for riociguat. Using homology modeling, small angle X-ray scattering (SAXS) and chemical cross-linking, we previously determined that Ms sGC lacking the cyclase domains (Ms sGC-NT) is an elongated molecule with a central parallel coiled-coil.…”