Amphipathic alpha-helix mediates the heterodimerization of soluble guanylyl cyclase.
Soluble guanylyl cyclase (soluble GC) is an enzyme consisting of alpha and beta subunits and catalyzes the conversion of GTP to cGMP. The formation of the heterodimer is essential for the activity of soluble GC. Each subunit of soluble GC has been shown to comprize three functionally different parts: a C-terminal catalytic domain, a central dimerization domain, and an N-terminal regulatory domain. The central dimerization domain of the beta(1) subunit, which contains an N-terminal binding site (NBS) and a C-terminal binding site (CBS), has been postulated to be responsible for the formation of alpha/ beta heterodimer. In this study, we analyzed heterodimerization by the pull-down assay using the affinity between a histidine tag and Ni(2+) Sepharose after co-expression of various N- and C-terminally truncated FLAG-tagged mutants of the alpha(1) subunit and the histidine-tagged wild type of the beta(1) subunit in the vaculovirus/Sf9 system, and demonstrated that the CBS-like sequence of the alpha(1) subunit is critical for the formation of the heterodimer with the beta(1) subunit and the NBS-like sequence of the alpha(1) subunit is essential for the formation of the enzymatically active heterodimer, although this particular sequence was not involved in heterodimerization. The analysis of the secondary structure of the alpha(1) subunit predicted the existence of an amphipathic alpha-helix in residues 431-464. Experiments with site-directed alpha(1) subunit mutant proteins demonstrated that the amphipathicity of the alpha-helix is important for the formation of the heterodimer, and Leu(463) in the alpha-helix region plays a critical role in the formation of a properly arranged active center in the dimer.
Shiga
Takumi
T
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Japan.
Suzuki
Norio
N
D000595:Amino Acid Sequence; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D000071756:Soluble Guanylyl Cyclase
D021122:Protein Subunits; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase