Structural Determinants for the Activation of Soluble Guanylyl Cyclase

Abstract

Soluble guanylyl cyclase (GC-1) converts guanosine 5'-triphosphate (GTP) into cyclic guanosine 3',5'-monophosphate (cGMP), a potent vasodilator. Nitric oxide (NO) binds to an N-terminal heme cofactor and stimulates GC-1 activity 100 – 200-fold, inducing vasodilation. In cardiovascular diseases, GC-1 heme is prone to oxidation which abolishes NO-sensitivity and reduces cGMP turnover, causing vasoconstriction and enhancing oxidative stress. Because GC-1 is central to the pathway regulating vascular function, the enzyme is a target for pharmaceutical intervention to promote GC-1 activity and improve cardiovascular health. However, the structural elements that relay the NO-binding event to the catalytic domain are unknown. We hypothesize that a series of amino acids promotes GC-1 catalytic activity upon NO binding through a network of hydrogen bonds and hydrophobic interactions. Identification of these amino acids will aid structure-based drug design efforts to target dysfunctional GC-1. To identify these amino acids, we designed, optimized, and tested a luciferase reporter assay, which relies on a cGMP-dependent promoter upstream of the luciferase gene. In an E. coli host, GC-1 was expressed and produced cGMP which induced luciferase expression. Luciferase activity was measured in cell lysates as an indirect measurement of GC-1 activity. We optimized this assay by using cAMP-deficient E. coli cells to decrease background luciferase activity by ~90%. By targeting distinct structural elements in the catalytic domain through site-directed mutagenesis, we identified several novel activating GC-1 variants and confirmed their effect via extracellular cGMP measurements. When mutants from various regions are combined, we measured synergistic or antagonistic effects, supporting our hypothesis whereby discrete regions in GC-1 act as hotspots for allosteric regulation of GC-1 activity. Our lab had previously determined the structure of the apo inactive wild-type heterodimeric catalytic domain of GC-1 (??GCcat). To determine a structure of the active ??GCcat conformation and to overcome preferential ??GCcat homodimerization, we made several mutants and constructs of the catalytic domain. However, we only crystallized and solved the structure of mutant ??GCcat. Overall, our results describe the design of the first bacterial assay to identify activating GC-1 mutations, and suggest cross talk between various structural elements of the catalytic domain, which regulate GC-1 activity.