Fig. 1. The structure of Arl2-GTP. (A) Electron density maps around the bound nucleotide [2F_o – F_c map contoured at 1.8σ (green), F_o – F_c at 3σ (blue), and F_o – F_c at –3σ (red)]. Upper panel: GTP, Mg²⁺ (yellow sphere) and a water molecule (W13) in the active site of Arl2 in crystal form-1, at 2.3 Å resolution. Lower panel: GDP, phosphate and Mg²⁺ in the active site of Arl2 in form-2, at 1.8 Å resolution. Note the positive difference density between the β phosphate and the phosphate ion, and, on the right, negative density at the phosphate ion and positive density for an additional water not added to the current model, indicating only partial hydrolysis and the presence of residual GTP/water. (B) Ribbon diagram of Arl3-GDP (with the additional β-strand β_2E) and Arl2-GTP (form-1), with switches in blue, interswitch region in green, N-terminus in yellow, C-terminal helix in magenta, nucleotides in orange and Mg²⁺ as red sphere. (C) Sequence alignment of mmArl2, mmArl3 and hArf1. Contact sites between Arl2 and PDEδ are highlighted in red. Color coding in the amphipathic N-terminal region: hydrophobic residues in yellow, basic residues in blue. Phenylalanines characteristic for Arf proteins are boxed; conserved residues are in bold. Secondary structure elements were determined by PROCHECK (CCP4, 1994) (D) Schematic representation of the observed β register shift resulting in the release of the N-terminus. Color coding as in (B).

Fig. 2. The structure of PDEδ and comparison with RhoGDI. (A) Ribbon representations of PDEδ (crystal form-1 and -2) and RhoGDI (Gosser et al., 1997). Disordered loops are shown as dotted lines. Note the degeneration of secondary structure and higher disorder in form-1, marked by an ellipse. (B) Structure based sequence alignment of full-length PDEδ (crystal form-2) and the C-terminal domain of RhoGDI1 (residues 59–204) produced with BRAGI (Schomburg and Reichelt, 1988). Highlighted in yellow are those residues of RhoGDI that contribute to the lipid binding pocket in the structure of Cdc42-GDP:RhoGDI (Hoffman et al., 2000), and structurally equivalent residues that line the inner surface of the hydrophobic pocket of PDEδ. Residues contributing to the interface with Cdc42-GDP and Arl2, respectively, are highlighted in red.

Fig. 3. Overall view of Arl2-GTP:PDEδ and comparison with Rap1A-GppNHp:RafRBD and Cdc42-GDP:RhoGDI. (A) Ribbon diagram of the complex of Arl2-GTP and PDEδ, with color coding of Arl2 as in Figure 1B. (B) Complex of Rap1A-GppNHp and the Ras binding domain of Raf (Nassar et al., 1995). Color coding as in (A). (C) Complex of Cdc42-GDP and RhoGDI1 (Hoffman et al., 2000). Color coding as in (A). C-terminal geranylgeranyl modification is shown in light blue.

Fig. 4. The interface of Arl2 and PDEδ. (A) Stereo representation of a view along the β–β interface (Arl2-β2 and PDEδ-β7). (B) Schematic representation. Color coding as in Figure 3. Interactions as dotted lines. Residues are boxed, water molecules are represented by circles.

Fig. 5. The hydrophobic pocket in PDEδ. (A) Cut through a surface representation of RhoGDI showing the deep hydrophobic pocket with the geranylgeranyl moiety from the C-terminus of Cdc42-GDP (GDI is shown as a violet ribbon; geranylgeranyl moiety is shown in orange in stick representation). (B) PDEδ features a less deep but similarly hydrophobic pocket. The geranylgeranyl moiety of Cdc42 has been positioned by superimposition of the protein backbones of RhoGDI to PDEδ, followed by a small manual adjustment within the pocket. Full opening of the pocket would require only a small conformational change.

Fig. 6. Identification of new targets of PDEδ and initial characterization of the interaction between PDEδ and H-Ras. (A) Qualitative two-hybrid analysis. Growth data reveal H-Ras, Rheb, Rho6 and Gα_i1 as new interaction partners of PDEδ. (B) Quantitative β-galactosidase assay of the interactors shown in (A). (C) Quantitative β-galactosidase assay of PDEδ with the indicated mutant forms of H-Ras (3CS = C181S, C184S, C186S).