Structural and Functional Characterization of the Dimerization Sites of Soluble Guanylyl Cyclase

Soluble guanylyl cyclase (sGC) is a ubiquitous enzyme that functions as a receptor for nitric oxide. In spite of the obligate heterodimeric nature of sGC, the sequence mediating subunit association have remained elusive. Our initial screening for the interaction site(s) demonstrated that two regions of each subunit, i.e. the regulatory domain and the central region, are involved in heterodimer formation of the most common sGC isoenzyme, α ₁/β₁. To precisely map the relevant segments in the β₁ subunit, we constructed multiple N- and C-terminal deletion variants and co-transfected them with full-length α₁ in COS cells. Immunoprecipitation revealed that a sequence segment spanning positions 204 to 408 mediates binding of α₁ to β₁. Fusion of the α₁ dimerization region to EGFP conferred binding activity to the recipient protein. Analysis of deletion constructs lacking portions of the dimerization region identified two distinct sites in β ₁ that contribute importantly to its interaction with α ₁, i.e. an N-terminal binding site (NBS) covering positions 204 to 244 and a C-terminal binding site at 379 to 408 (CBS). Both NBS and CBS are crucial for sGC function as deletion of either site rendered sGC inactive. We conclude that the dimerization region of β₁ extends over 205 residues of its regulatory and central domains, and that two discontinuous sites of 41 and 30 residues, respectively facilitate binding of β ₁ to the α₁ subunit of sGC.