Structure of the GDP-bound G domain of the RGK protein Rem2

Abstract

RGK proteins are atypical small GTP-binding proteins that are involved in the regulation of voltage-dependent calcium channels and actin cytoskeleton remodelling. The structure of the Rem2 G domain bound to GDP is reported here in a monoclinic crystal form at 2.66 Å resolution. It is very similar to the structure determined previously from an orthorhombic crystal form. However, differences in the crystal-packing environment revealed that the switch I and switch II regions are flexible and not ordered as previously reported. Comparison of the available RGK protein structures along with those of other small GTP-binding proteins highlights two structural features characteristic of this atypical family and suggests that the conserved tryptophan residue in the DXWEX motif may be a structural determinant of the nucleotide-binding affinity.

Figure 1

Structural characterization of the G domain of Rem2 bound to GDP. (a) Structure of the monoclinic crystal form. The N- and C-termini of switches I and II are shown in red and green, respectively. (b) SEC-MALLS analysis of Rem2_Gdomain–GDP at 29 mg ml⁻¹. The size-exclusion profiles of the protein (monitored by refractometry) and the molecular masses (calculated from light-scattering and refractometry data) are plotted. (c) A cross-eye stereoview of the superposition of Rem2–GDP in the monoclinic (black, this study) and orthorhombic (blue, PDB entry 3cbq) crystal forms. Note the well defined conformation of switches I and II in the orthorhombic form.

Figure 2

Sequence alignment based on crystal structures of the G domains of RGK proteins. Human Gem, Rad and Rem1 and rat Rem2 sequences were used.

Figure 3

Structural features of the GDP-bound G domains of the RGK proteins. (a) Superposition between the H-Ras protein (PDB entry 4q21; Milburn et al., 1990; shown in black) and the four RGK proteins (shown in colours). Gem (PDB entry 2cjw), Rad (PDB entry 2dpx), Rem1 (2nzj) and Rem2 (PDB entry 3cbq) are shown in orange, violet, blue and green, respectively. The residues at positions 2 and 3 of the G3 motif (DTAGQ in H-Ras and DXWEX in RGK) are indicated in stick representation. Note that human sequence numbering is used for Rem2. (b) A close-up view of the G3 motif of Gem (PDB entry 2cjw; only one RGK protein is indicated for clarity) compared with H-Ras (PDB entry 4q21) is shown with residues at positions 2 and 3 indicated in stick representation. (c) The hydrophobic pocket surrounding the conserved G3 motif tryptophan. A superposition of the four RGK proteins is shown as defined in (a). (d) A close-up view of the nucleotide pocket of H-Ras (PDB entry 4q21) is shown with Ala59 from the G3 motif modelled as a tryptophan. For comparison, Gem (PDB entry 2cjw; only one RGK protein is indicated for clarity) is shown superposed with H-Ras. The grey area indicates the steric hindrance between the modelled tryptophan of H-Ras and the water-coordination shell of the magnesium ion