Structural and biochemical investigations into the mechanism and evolution of soluble guanylate cyclase regulation

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Project summary Nitric oxide (NO) is an intercellular signaling molecule used across several kingdoms of life, and in animals, NO is involved in key processes like vasodilation, neurotransmission, and the host response to infection. Central to NO function in animal physiology is the metalloenzyme soluble guanylate cyclase (sGC), which generates the secondary messenger guanosine 3’,5’-monophosphate (cGMP) in response to NO1,2. Because of its centrality to several physiological processes in humans, sGC is a current therapeutic target3, and understanding the struc- tural underpinnings of sGC activation will inform continued development. Though NO has only been proven to bind at the heme cofactor, sGC activity has three distinct stages: 1) low basal activity without NO bound (NO- free), 2) moderate activity (10-fold increase) with a single, heme-bound NO (1-NO), and 3) maximal activity (200- fold increase) with excess NO4. Biochemical5 and structural6,7 experiments suggest NO allosterically regulates sGC activation through elongation of a coiled-coil domain between the N-terminal NO-binding regulatory domain and the C-terminal catalytic domain. However, interdomain arrangement and the conformational population dis- tribution of the physiologically relevant 1-NO state remain to be fully explored. While metazoan sGCs exist as heterodimers with only one heme-binding domain, the recently discovered single-celled eukaryotic Choanoeca flexa (Cf) sGC is a homodimer that nonetheless exhibits half-sites heme-loading and 3-stage NO-activation8,9. Through this proposed research, we will probe how Cf sGC allosterically modulates activity in response to NO stimulation. Activity assays, homology-guided mutational studies, and a survey of small molecule stimulators will be used to characterize Cf sGC activation. Cryo-electron microscopy will be used to determine high-resolution structures of Cf sGC in active and inactive states, allowing for visualization of key structural similarities and differences between Cf sGC and animal counterparts. Small-angle X-ray scattering (SAXS) experiments will be performed with both Cf sGC and animal sGCs to deconvolve the putative 3-stage NO-activation profile of sGC. These experiments aim to identify and describe the structural states of Cf sGC and illuminate the structural differences between ancestral and animal sGCs that define the evolutionary history of NO-based signaling.