Double-mutant analysis of the interaction of Ras with the Ras-binding domain of RGL

RalGDS is a guanine nucleotide dissociation stimulator for Ral, and one of its homologues is RGL (RalGDS-like). In this study, the effects of mutation s of Ras and the Ras-binding domains (RBDs) of RalGDS and RGL on their bind ing have been systematically examined. The D33A mutation of Ras reduces the abilities to bind RGL-RBD and RalGDS-RBD. To identify the RGL residue inte racting with Asp33 of Ras, double-mutant analyses between Ras and RGL-RBD w ere conducted. For example, the K685A mutation of RGL-RBD has a much smalle r effect on the RGL-RBD binding ability of the D33A mutant than on those of other mutants of Ras. Accordingly, it is indicated that the attractive int eraction of Asp33 in Ras with Lys685 in RGL-RBD (Lys816 in RalGDS-RBD) cont ributes to the Ras.RBD association. This interaction is consistent with the crystal structure of the complex of RalGDS-RBD and the E31K Ras mutant [Hu ang, L., Hofer, F., Martin, G. S., and Kim, S.-H. (1998) Not. Struct. Biol. 5, 422-426]. This crystal structure exhibits interactions of the muration- derived Lys31 side chain with three RalGDS residues. Glu31 of Ras discrimin ates Ras from a Ras-homologue, Rap1, with Lys31, with respect to RalGDS and RGL binding; the E31K mutation of Res potentiates the abilities to bind RG L-RBD and RalGDS-RBD. To examine the role of Glu31 of the wild-type Ras in the interaction with RGL and RalGDS, double-mutant analyses were conducted. The Ras binding ability of the E689A mutant of RGL-RBD is much stronger th an that of the wild-type RGL-RBD, and the E31K mutation of Ras no longer po tentiates the Ras binding ability of the E689A mutant. Therefore, the repul sive interaction between Glu31 in Ras and Glu689 in RGL-RBD (Asp820 in RalG DS-RBD) may keep the Ras.RBD association weaker than the Rap1.RBD associati on, which might be relevant to the regulation of the signaling network.