Structural basis of membrane targeting by the Dock180 family of Rho family guanine exchange factors (Rho-GEFs)

Abstract

The Dock180 family of atypical Rho family guanine nucleotide exchange factors (Rho-GEFs) regulate a variety of processes involving cellular or subcellular polarization, including cell migration and phagocytosis. Each contains a Dock homology region-1 (DHR-1) domain that is required to localize its GEF activity to a specific membrane compartment where levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) are up-regulated by the local activity of PtdIns 3-kinase. Here we define the structural and energetic bases of phosphoinositide specificity by the DHR-1 domain of Dock1 (a GEF for Rac1), and show that DHR-1 utilizes a C2 domain scaffold and surface loops to create a basic pocket on its upper surface for recognition of the PtdIns(3,4,5)P(3) head group. The pocket has many of the characteristics of those observed in pleckstrin homology domains. We show that point mutations in the pocket that abolish phospholipid binding in vitro ablate the ability of Dock1 to induce cell polarization, and propose a model that brings together recent mechanistic and structural studies to rationalize the central role of DHR-1 in dynamic membrane targeting of the Rho-GEF activity of Dock180.

FIGURE 1.

Structure of the Dock1 DHR-1 domain. A, domain organization of Dock1, with residue limits and major binding partners indicated. ELMO1 binds to a module comprising the SH3 domains and “EB” helical segment, as well as segments at the C terminus (not shown) (11, 85). ARM, Armadillo/HEAT repeat; EB, ELMO-binding; PL, phospholipid; Crk, CrkII. B, stereo Cα plot of DHR-1 (cyan) overlaid with the C2 domain of PtdIns 3-kinase-γ (red). The latter is disordered at loop L3 and has shorter β2–β3 and β7–β8 insertions. DHR-1 is disordered between residues 579 and 588. Strands and loops are numbered, and termini labeled. C, schematic of DHR-1 in the same view as in B. The β-sandwich core (with strands numbered) is in green, insertions in blue. D, surface charge potential (83) on DHR-1. The view is rotated ∼90° about a vertical axis to show the basic surfaces, with computational docking solutions (see text) for the PtdIns(3,4,5)P₃ head group in the upper surface pocket and β-groove (note that there is no experimental evidence for binding in the β-groove).

FIGURE 2.

Sequences of Dock180 family DHR-1 domains. A, domain organization of the 4 groups of human full-length proteins. B, structure-based alignment of the 11 human DHR-1 sequences and Drosophila myoblast city, which belongs to the DockA group. The secondary structure is indicated. The 3 surface loops, L1–L3, are indicated by red boxes; they are delimited by conserved residues marked by black arrows. Residues predicted to contact phospholipid in Dock1 DHR-1 are marked with a red asterisk; those supported by mutagenesis are boxed in blue. Contact residues in other family members predicted by homology are shown in blue (H/K/R) or red (others). Conserved structural residues are in black (bold). Residues mutated in this study that do not affect phospholipid binding are highlighted in yellow; 3 of these are on L3 but point into the β-groove. The 2 large insertions in the C2 core are also indicated.

FIGURE 3.

Phosphoinositide binding to the DHR-1 domain. A, the head group of PtdIns(3,4,5)P₃ with phosphate positions numbered. B, thermodynamic parameters for phosphoinositide and head group binding to wild-type DHR-1 (K_d, dissociation constant; ΔH, enthalpy change; TΔS, temperature (K) × entropy change; ΔG (the free energy change) = ΔH-TΔS = −RT ln K_d); N is the apparent stoichiometry. No binding was observed for PtdIns(3,5)P₂, PtdIns(3,4)P₂, or PtdIns(3)P₁. * indicates the experiment was performed in 145 mm NaCl. All other experiments were carried out in low salt (see “Experimental Procedures”). Representative ITC profiles are provided in supplemental Fig. S4. C, effect of point mutations on PtdIns(3,4,5)P₃ binding, defined as K_d(wt)/K_d(mutant). Error estimates are from fitting of ITC titration curves. At right are melting temperatures, T_m, for each mutant, in °C. *, the T_m value given for K524A is actually for the triple mutant, K446A/K524A/K555A. D, mutation sites mapped onto the DHR-1 structure. E and F, ITC competition titrations (negative values of energy indicate an exothermic reaction; positive values, endothermic). E, DHR-1 equilibrated with a 1.5 m excess of PtdIns(4,5)P₂ titrated with PtdIns(3,4,5)P₃. F, the converse experiment: DHR-1/PtdIns(3,4,5)P₃ titrated with PtdIns(4,5)P₂.

FIGURE 4.

Point mutations in the DHR-1 domain inhibit cell elongation by Dock1. A, cells transfected with plasmids expressing wild-type or mutant Dock1, together with plasmids for ELMO1 and CrkII, were detached and plated on fibronectin-coated chambers for 2 h, and stained with anti-Dock1 antibody, rhodamine, phalloidin, and 4,6-diamidino-2-phenylindole (photographed at ×100 magnification: scale bar, 10 μm). B, quantification of cell behavior. Independent fields were photographed at lower magnification, and scored for three phenotypes: round, spread, and elongated. Data in A and B are representative of three independent experiments. C, expression levels of transfected proteins analyzed by immunoblotting cell lysates.

FIGURE 5.

The PtdIns(3,4,5)P₃ binding pocket in DHR-1 and comparison with ARNO. A, stereo image of the PtdIns(3,4,5)P₃ head group docked into the upper surface pocket of the DHR-1 domain, with a zoom-in to show residues predicted to form the pocket and/or bind phospholipid. B, same view as the zoom-in with the binding pocket shown as electrostatic surface. C and D, ARNO PH domain and its complex with Ins(1,3,4,5)P₄ (PDB code 1U27) (60), with the pocket oriented to show surface similarity.

FIGURE 6.

Surface conservation of Dock180 proteins. Sequence conservation within the DockA/B group (green, conserved; purple, variable) of DHR-1 mapped onto Dock1 DHR-1. 3 views are shown and compared with the electrostatic surface potential. A is a view along the edge of the β-sandwich; B is a side view showing the β-groove; and C is a top view looking down onto the upper surface, with InsP₄ docked into the upper surface binding pocket.

FIGURE 7.

Hypothetical model of the dimeric Dock1-Rac1 membrane complex. A, the predicted interdomain HEAT/ARM repeats (see also supplemental Figs. S9 and S10) are shown as orange molecular surfaces, onto which DHR-1 (blue ribbon) is modeled by analogy with the structure of PtdIns 3-kinase (84). The DHR2 dimer (green/lime surface) bound to Rac1 (magenta ribbon) is modeled on the Dock9-Cdc42 complex (13). Membrane attachment sites for DHR-1 (PtdIns(3,4,5)P₃ head group) and Rac-1 (prenylated C terminus) are indicated. B, the same as A but rotated by 90° about a horizontal axis, providing a “membrane view” of the complex. The region N-terminal to the DHR-1 domain (shown schematically) is predicted to lie adjacent to the DHR-2 domain; it includes the SH3 domain in DockA/B or the PH domain in DockD.