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1 July 2005 Amphipathic α-Helix Mediates the Heterodimerization of Soluble Guanylyl Cyclase
Takumi Shiga, Norio Suzuki
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Soluble guanylyl cyclase (soluble GC) is an enzyme consisting of αand β 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 β1 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 α/β heterodimer. In this study, we analyzed heterodimerization by the pull-down assay using the affinity between a histidine tag and Ni2 Sepharose after co-expression of various N- and C-terminally truncated FLAG-tagged mutants of the α1 subunit and the histidine-tagged wild type of the β1 subunit in the vaculovirus/Sf9 system, and demonstrated that the CBS-like sequence of the α1 subunit is critical for the formation of the heterodimer with the β1 subunit and the NBS-like sequence of the α1 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 α1 subunit predicted the existence of an amphipathic α-helix in residues 431–464. Experiments with site-directed α1 subunit mutant proteins demonstrated that the amphipathicity of the α-helix is important for the formation of the heterodimer, and Leu463 in the α-helix region plays a critical role in the formation of a properly arranged active center in the dimer.

Takumi Shiga and Norio Suzuki "Amphipathic α-Helix Mediates the Heterodimerization of Soluble Guanylyl Cyclase," Zoological Science 22(7), 735-742, (1 July 2005).
Received: 30 March 2005; Accepted: 1 June 2005; Published: 1 July 2005
amphipathic α-helix
dimerization region
pull-down assay
recombinant protein
secondary structure
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