RGK small GTP-binding proteins interact with the nucleotide kinase domain of Ca2+-channel β-subunits via an uncommon effector binding domain

RGK proteins (Kir/Gem, Rad, Rem, and Rem2) form a small subfamily of the Ras superfamily. Despite a conserved GTP binding core domain, several differences suggest that structure, mechanism of action, and functional regulation differ from Ras. RGK proteins down-regulate voltage-gated calcium channel activity by binding in a GTP-dependent fashion to the Ca-v beta subunits. Mutational analysis combined with homology modeling reveal a novel effector binding mechanism distinct from that of other Ras GTPases. In this model the Switch 1 region acts as an allosteric activator that facilitates electrostatic interactions between Arg-196 in Kir/Gem and Asp-194, -270, and -272 in the nucleotide-kinase (NK) domain of Ca-v beta 3 and wedging Val-223 and His-225 of Kir/ Gem into a hydrophobic pocket in the NK domain. Kir/ Gem interacts with a surface on the NK domain that is distinct from the groove where the voltage-gated calcium channel Ca-v beta 1 subunit binds. A complex composed of the RGK protein and the Ca-v beta 3 and Cav1.2 subunits could be revealed in vivo using coimmunoprecipitation experiments. Intriguingly, docking of the RGK protein to the NK domain of the Ca-v beta subunit is reminiscent of the binding of GMP to guanylate kinase.