UC Berkeley

Nitric oxide (NO) is an indispensable gas signaling molecule and immune system effector in mammals. In the prototypical NO signaling pathway that mediates vasodilation in humans, the source of NO is a nitric oxide synthase (NOS), and NO is sensed by the heme-containing gas sensor soluble guanylate cyclase (sGC). Mammalian sGCs are α/β-type heterodimers activated by NO and catalyze the conversion of GTP to the secondary messenger cyclic GMP (cGMP). sGCs regulate critical physiological functions such as vasodilation and neuronal signaling, and misregulation of sGC leads to diseases. Therefore, sGC activity regulation has been a target of significant research effort. A major step towards elucidating the mechanism of mammalian sGC activation came about in 2018, when the cryo-electron microscopy structure of human sGC and an insect homolog of sGC from tobacco hornworm, Manduca sexta, were reported. This, combined with increasingly robust structural prediction techniques, greatly facilitated mechanistic studies of sGC and provided new tools to study diverse non-mammalian homologs of sGC proteins.

Besides mammals, NO signaling is important for a wide range of organisms including bacteria, algae, fungi, and many invertebrate animals. Animal pathways that are frequently regulated by NO include larval metamorphosis, flagellar movement and collective contractility, to name a few. From an evolutionary standpoint, choanoflagellates are an especially interesting organism to study NO signaling, as they are the closest living relatives of animals and may hold the key to understanding the evolution of NO signaling. An in vivo study described here established the presence of a NO signaling pathway in the colonial choanoflagellate Choanoeca flexa. C. flexa co-expresses NOS and sGC. NO stimulates sGC activity in C. flexa and drives the collective contraction behavior. cGMP produced by sGC is necessary for persistent contraction of C. flexa colonies. These data provided insight into a potential role of NO in early animals and evolution of NO signaling, and brought a NO-sensitive sGC, C. flexa sGC1 into the spotlight. Initial characterization of Cf sGC1 in vitro demonstrated that it is a catalytically active homodimeric protein. Ligand binding properties and the ligand-induced activity profile of Cf sGC1 are both reminiscent of α/β-type sGC. These data prompted a more detailed biochemical characterization of Cf sGC1. Besides additional similarities in substrate kinetics, structural studies using small angle X-ray scattering revealed that Cf sGC1 is a homodimer with conformational asymmetry. This asymmetry may be connected to the 1-heme per homodimer heme stoichiometry of Cf sGC1, also determined in this study. Furthermore, like α/β-type sGC, a conformational extension was observed for Cf sGC1 during activation. Similarities between Cf sGC1 and α/β-type sGC suggest comparable mechanisms of activity regulation.

Like animal sGCs, Cf sGC1 displays a three-stage activation profile, suggesting that NO binding to heme alone is insufficient for full activation. In α/β-type sGC, cysteines were hypothesized to mediate secondary NO interaction. Involvement of cysteines in controlling the activity of Cf sGC1 was tested. Cf sGC1 treated with the cysteine labeling reagent methyl methanethiosulfonate (MMTS) was inhibited, suggesting that MMTS treatment either blocked non-heme NO interaction, or blocked conformational change at a critical cysteine site. Cysteine variants of Cf sGC1 at conserved sites to α/β-type sGC were prepared and characterized. Unexpectedly, cysteine variants of Cf sGC1 did not exhibit different properties in heme ligand binding, ligand-induced activation or MMTS-mediated inhibition. Additional work is required to definitively show the link of cysteines to activity regulation in Cf sGC1.

Under aerobic conditions, α/β-type sGCs and Cf sGC1 do not bind O2. The study of ligand selectivity in α/β-type sGCs led to the discovery of sGC that can bind O2. However, the mechanism for activity regulation of O2-binding sGCs is not well understood. Besides Cf sGC1, the genome of C. flexa also encodes sGCs that bind O2. Here, the O2-binding sGC, Cf sGC4 was characterized using biochemical and structural techniques. Unlike homologs that bind NO selectively, Cf sGC4 is inhibited by ligand binding, including NO, CO and O2, and did not undergo conformational change in the presence of NO. These results support a different and currently unknown mechanism of activity regulation in Cf sGC4 and possibly other O2-binding sGCs.