YC-1 Binding to the β Subunit of Soluble Guanylyl Cyclase Overcomes Allosteric Inhibition by the α Subunit

Abstract

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remains unknown. Using linked-equilibria binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the beta subunit. In the absence of CO, YC-1 binds with Kd = 9–21 μM, depending on construct. In the presence of CO, these values decrease to 0.6–1.1 μM. Pfizer compound 25 bound ~10-fold weaker than YC-1 in the absence of CO whereas compound BAY 41–2272 bound particularly tightly in the presence of CO (Kd = 30–90 nM). Additionally, we found that CO binding is much weaker to heterodimeric sGC proteins (Kd = 50–100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca beta H-NOX/PAS). YC-1 greatly enhanced CO binding to heterodimeric sGC, as expected (Kd = ~1 μM). These data indicate the alpha subunit induces a heme pocket conformation with lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the alpha subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity.

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

“…Interestingly, BAY 41-2272 (10 µM) did not enhance CO binding to any of the four bacterial H-NOX proteins (supplemental Table S2). This may be due to the weaker compound binding but is also consistent with previous results with sGC, where stimulator enhanced CO binding to the heterodimeric protein, but not to the isolated H-NOX domain (20).…”

“…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).…”

“…S2). Heme was retained after 15 minutes of UV irradiation, as indicated by a minimal decrease in Soret band absorption and a slight shift in Soret maxima characteristic of stimulator binding (20) (supplemental Fig. S3).…”

“…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.…”

“…Though conserved, IWP-051 binding to Sw H-NOX is weaker than to Ms sGC-NT, with K d = 1.9 mM estimated for the CO complex by chemical shift perturbation NMR titration (Fig. 5B, supplemental Table S6) compared with K d values of 0.03-3.8 µM for various stimulator compounds binding to the CO complexes of Ms sGC-NT constructs (20).…”