A STABLE ALPHA-HELICAL DOMAIN AT THE N-TERMINUS OF THE RI-ALPHA SUBUNITS OF CAMP-DEPENDENT PROTEIN-KINASE IS A NOVEL DIMERIZATION DOCKING MOTIF/

The RI alpha subunit of cAMP-dependent protein kinase is maintained as an asymmetric dimer by a dimerization motif at the N terminus, Based on resistance to proteolysis and expression as a discrete domain in Es cherichia coil, this motif is defined as residues 12-61. This motif is chemically, kinetically, and thermally stable, The two endogenous int erchain disulfide bonds between Cys(16) and Cys(37) in RI alpha are ex tremely resistant to reduction even in 8 M urea, indicating that they are well shielded from the reducing environment of the cell. The disul fide bonds were present in recombinant RI alpha as well as when the di merization domain alone was expressed in E. coil, emphasizing the unus ual stability of this motif and the disulfide bonds. Although 100 mM, dithiothreitol was sufficient to reduce the disulfide bonds, it did no t abolish dimerization. In addition, a stable dimer also still formed when Cys(37) was replaced with His, confirming unambiguously the origi nal antiparallel alignment of the disulfide bonds, Thus, both in vitro and in vivo, disulfide bonds are not required for dimerization. Circu lar dichroism of the dimerization domain indicated a high content of a thermostable alpha-helix. Based on the CD data, trypsin resistance of the fragment, location of the disulfide bonds, and amphipathic helix predictions, potential models are discussed, A new alignment of the di merization domains of RI, RII, and cGMP-dependent protein kinase eluci dates fundamental similarities as well as significant differences amon g these three domains.