Efficient expression of human soluble guanylate cyclase in Escherichia coli and its signaling-related interaction with nitric oxide.
Soluble guanylate cyclase (sGC), as a nitric oxide (NO) sensor, is a critical heme-containing enzyme in NO-signaling pathway of eukaryotes. Human sGC is a heterodimeric hemoprotein, composed of a alpha-subunit (690 AA) and a heme-binding beta-subunit (619 AA). Upon NO binding, sGC catalyzes the conversion of guanosine 5'-triphosphate (GTP) to 3',5'-cyclic guanosine monophosphate (cGMP). cGMP is a second messenger and initiates the nitric oxide signaling, triggering vasodilatation, smooth muscle relaxation, platelet aggregation, and neuronal transmission etc. The breakthrough of the bottle neck problem for sGC-mediated NO singling was made in this study. The recombinant human sGC beta1 subunit (HsGC beta 619) and its truncated N-terminal fragments (HsGC beta 195 and HsGC beta 384) were efficiently expressed in Escherichia coli and purified successfully in quantities. The three proteins in different forms (ferric, ferrous, NO-bound, CO-bound) were characterized by UV-vis and EPR spectroscopy. The homology structure model of the human sGC heme domain was constructed, and the mechanism for NO binding to sGC was proposed. The EPR spectra showed a characteristic of five-coordinated heme-nitrosyl species with triplet hyperfine splitting of NO. The interaction between NO and sGC was investigated and the schematic mechanism was proposed. This study provides new insights into the structure and NO-binding of human sGC. Furthermore, the efficient expression system of E. coli will be beneficial to the further studies on structure and activation mechanism of human sGC.
Zhong
Fangfang
F
Department of Chemistry, Fudan University, 200433, Shanghai, China.
Wang
Hongyan
H
Ying
Tianlei
T
Huang
Zhong-Xian
ZX
Tan
Xiangshi
X
D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D006162:Guanylate Cyclase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009569:Nitric Oxide / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D000071756:Soluble Guanylyl Cyclase; D013056:Spectrophotometry, Ultraviolet
D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase