Abstract
Soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. NO binds to the heme of sGC to catalyze the synthesis of the second messenger cGMP, which plays a critical role in several physiological processes. However, the molecular mechanism for sGC to mediate the NO signaling remains unclear. Here fluorophore FlAsH-EDT2 and fluorescent proteins were employed to study the NO-induced sGC activation. FlAsH-EDT2 labeling study revealed that NO binding to the H-NOX domain of sGC increased the distance between H-NOX and PAS domain and the separation between H-NOX and coiled-coil domain. The heme pocket conformation changed from “closed” to “open” upon NO binding. In addition, the NO-induced conformational change of sGC was firstly investigated in vivo through fluorescence lifetime imaging microscopy. The results both in vitro and in vivo indicated the conformational change of the catalytic domain of sGC from “open” to “closed” upon NO binding. NO binding to the heme of H-NOX domain caused breaking of Fe-N coordination bond, initiated the domain moving and conformational change, induced the allosteric effect of sGC to trigger the NO-signaling from H-NOX via PAS & coiled-coil to the catalytic domain, and ultimately stimulates the cyclase activity of sGC.
Highlights
The diatomic gas nitric oxide (NO) is an essential signaling molecule in biology
For the emission spectrum of FlAsH-EDT2 has good overlap with α and β absorbance bands of the heme, the FlAsH-EDT2 is a good probe to study the conformational change induced by NO binding based on the energy transfer between heme and FlAsH-EDT2
Previous work has been confirmed that the replacement of the residues (TC) and the FlAsH-EDT2 would not affect the heme microenviroment and heme binding in the Soluble guanylate cyclase (sGC) β1 variants [sGC β1(1-385)-386TC391, sGC β1(1-385)-243TC248 and sGC β1(1-619)-243TC248insect]31
Summary
Cyclase Activation by NO in vitro received: 06 September 2016 accepted: 19 January 2017. Several models have been proposed to clarify the mechanism of NO-induced sGC activation using a variety of techniques, including mutational and truncation studies, FRET and HDX-MS in vitro[21,23,24,25,26] Both these models revealed the inter-domain interactions and the conformational change upon NO binding. Firstly a fluorophore FlAsH-EDT2, as a conformational change indicator, was introduced into the truncated and full-length sGC β1 subunits to study the NO-induced conformational change of sGC in vitro, which revealed that NO binding could increase the distance between the H-NOX and PAS domain and the distance between H-NOX and coiled-coil domain. The conformational allosteric effect induced the NO-signaling from H-NOX domain to the catalytic domain to stimulate the cyclase activity of sGC
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