Abstract
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 Cavbeta 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 Cavbeta3 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 Cavalpha1 subunit binds. A complex composed of the RGK protein and the Cavbeta3 and Cav1.2 subunits could be revealed in vivo using coimmunoprecipitation experiments. Intriguingly, docking of the RGK protein to the NK domain of the Cavbeta subunit is reminiscent of the binding of GMP to guanylate kinase.
Highlights
Small GTPases comprise four major branches, Rab, Ras, Arf, and Rho
RGK proteins lack critical residues in the G2 (i.e. T35) and G3 (i.e. DXXG60) elements that are important for GTP hydrolysis in other Ras superfamily members [7]
Our data indicate that RGK proteins and the VDCC ␣-subunit bind to distinct surfaces on the nucleotide kinase (NK) domain of the -subunit and reveal an unusual effector binding mechanism for these small GTPbinding proteins
Summary
Small GTPases comprise four major branches, Rab, Ras, Arf, and Rho. The RGK3 family belongs to the Ras superfamily and includes Rad [1], Rem [2], Rem2 [3, 4], and Kir/Gem [5, 6]. At the position corresponding to Val-223, Rad, Rem, and Rem2 carry a Leu, and Kir/Gem V223L binds 3 with similar efficiency as WT Kir/ Gem. The side chains of either a Val or a Leu at this position fit into the hydrophobic pocket of Cav3, which is deep enough to accommodate the slightly longer side chain of a Leu or the bulkier one of a Trp. the side chain of a Met can insert into the hydrophobic pocket, its high degree of flexibility predicts a weaker interaction (supplemental Data VII), and the V223M substitution affects the association with Cav3 (see Fig. 3B, panel a, lane 8).
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