title: 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 remain unknown. Using linked equilibrium 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 β subunit. In the absence of CO, YC-1 binds with a Kd of 9-21 μM, depending on the 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 binds much more weakly to heterodimeric sGC proteins (Kd = 50-100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca β H-NOX/PAS). YC-1 greatly enhanced binding of CO to heterodimeric sGC, as expected (Kd ∼ 1 μM). These data indicate the α subunit induces a heme pocket conformation with a lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the α 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.
authors: Department of Chemistry and Biochemistry, The University of Arizona , Tucson, Arizona 85721, United States.
authors: Rahul
authors: R
authors: Purohit
authors: Bradley G
authors: BG
authors: Fritz
authors: Juliana
authors: J
authors: The
authors: Aaron
authors: A
authors: Issaian
authors: Andrzej
authors: A
authors: Weichsel
authors: Cynthia L
authors: CL
authors: David
authors: Eric
authors: E
authors: Campbell
authors: Andrew C
authors: AC
authors: Hausrath
authors: Leida
authors: L
authors: Rassouli-Taylor
authors: Elsa D
authors: ED
authors: Garcin
authors: Matthew J
authors: MJ
authors: Gage
authors: William R
authors: WR
authors: Montfort
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D002248:Carbon Monoxide / Q000737:chemistry; D002417:Cattle; D006162:Guanylate Cyclase / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D007191:Indazoles / Q000737:chemistry*; D018402:Manduca / Q000201:enzymology; D008958:Models, Molecular; D009569:Nitric Oxide / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D000071756:Soluble Guanylyl Cyclase; D020349:Surface Plasmon Resonance
chemical_list: D007191:Indazoles; D021122:Protein Subunits; D018160:Receptors, Cytoplasmic and Nuclear; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase