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. 2017 Feb 23:7:43112.
doi: 10.1038/srep43112.

Probing the Molecular Mechanism of Human Soluble Guanylate Cyclase Activation by NO in vitro and in vivo

Jie Pan  1 , Hong Yuan  1 , Xiaoxue Zhang  1 , Huijuan Zhang  1 , Qiming Xu  2 , Yajun Zhou  1 , Li Tan  3 , Shingo Nagawa  3 , Zhong-Xian Huang  1 , Xiangshi Tan  1   2
Affiliations

Probing the Molecular Mechanism of Human Soluble Guanylate Cyclase Activation by NO in vitro and in vivo

Jie Pan et al. Sci Rep. .

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.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Conformational change of FlAsH-labeled sGC β1(1-385)-243TC248, and sGC β1(1-385)-386TC391 upon NO binding (a and b) or CO binding (c and d). The heme concentration was 2 μM in 20 mM HEPES, 150 mM KCl, pH 7.4.
Figure 2
Figure 2
Anisotropy of FlAsH-labeled sGC β1(1-385)-243TC248 and sGC β1(1-385)-386TC391 upon NO binding (a) or CO binding (b). The heme concentration was 3 μM in 20 mM HEPES, 150 mM KCl, pH 7.4. Anisotropy data are the mean ± S.E. of two independent experiments performed in triplicate.
Figure 3
Figure 3
Conformational change of FlAsH-EDT2-labeled sGC β1(1-619)-243TC248 (a) and sGC β1(1-619)-386TC391 (b) upon NO binding. The heme concentration was 3 μM in 20 mM HEPES, 150 mM KCl, pH 7.4.
Figure 4
Figure 4
SRCD spectra in the far-UV region of sGC β1(1-385) (a) and sGC β1(1-385)-243TC248 (b) with and without DEA/NO. The heme concentration of sGC β1(1-385) and sGC β1(1-385)-243TC248 was 50 μM and 45 μM, respectively, in 10 mM HEPES, pH 7.4.
Figure 5
Figure 5
Electronic absorption spectra of sGC α1(1-690)-CFPinsect & β1(1-619)-YFPinsect (a) and sGC CFP-α1(1-690)insect & β1(1-619)-YFPinsect (b) before and after addition of DEA/NO and the mixture of DEA/NO and substrate GTP.
Figure 6
Figure 6. FRET analysis of purified sGC heterodimer.
Fluorescent spectra of sGC α1(1-690)-CFPinsect & β1(1-619)-YFPinsect (a) and sGC CFP-α1(1-690) insect & β1(1-619)-YFPinsect (b) at excitation wavelength of 432 nm before and after addition of DEA/NO and the mixture of DEA/NO and substrate GTP.
Figure 7
Figure 7. Co-immunoprecipitation analysis using transiently transfected SH-SY5Y cells expressing the wide type sGC α and β1 subunit or the sGC α and β1 mutant.
Cells were co-transfected with HA- and FLAG- tagged constructs, and the HA-tagged constructs were precipitated using anti-HA antibody. Co-precipitated FLAG-tagged constructs were detected by SDS-PAGE/immunoblotting using anti-FLAG antibody (top). As a control, precipitated HA-tagged constructs were detected using anti-HA antibody (bottom). The gels were cropped and the full-length gels were shown in Supplementary Fig. S3.
Figure 8
Figure 8. FLIM-FRET analysis in total cells.
The images were taken with SH-SY5Y cells transfected with sGC β1-CFP alone (a), sGC β1-CFP and YFP (b), sGC β1-CFP and sGC β1-YFP (c), sGC β1-CFP and sGC β1-YFP H105A (d), sGC β1-CFP and sGC β1-YFP after addition of DEA/NO (e), sGC β1-CFP and sGC β1-YFP after addition of DEA/NO and GTP (f), and sGC β1-CFP and sGC β1-YFP H105A after addition of DEA/NO (g). Confocal images show the cellular localization of α and β subunits. The rightmost images show the lifetime images. (h) The lifetime values were the average lifetime through fitting the decay curve to the double exponential function from FLIM images. (l) FLIM-FRET efficiency (E) values (mean ± S.E.) were calculated based on CFP lifetime values and obtained over 5–6 cells from 4 to 5 different samples.
Figure 9
Figure 9. FLIM-FRET analysis in total cells.
The images were taken with SH-SY5Y cells transfected with sGC α2-CFP alone (a), sGC α2-CFP and YFP (b), sGC α2-CFP and sGC β1-YFP (c), and sGC α2-CFP and sGC β1-YFP after addition of DEA/NO (d). Confocal images show the cellular localization of α and β subunits. The rightmost images show the lifetime images. (e) The lifetime values were the average lifetime through fitting the decay curve to the double exponential function from FLIM images. (f) FLIM-FRET efficiency (E) values (mean ± S.E.) were calculated based on CFP lifetime values and obtained over 5–6 cells from 4 to 5 different samples.
Figure 10
Figure 10. A proposed NO-Induced activation mechanism of sGC.
Figure 11
Figure 11. The domain architectures of sGC and schematic representations of sGC variants constructs used in this study.
See this image and copyright information in PMC

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