Multiple interactions between an Arf/GEF complex and charged lipids determine activation kinetics on the membrane

Abstract

Lipidated small GTPases and their regulators need to bind to membranes to propagate actions in the cell, but an integrated understanding of how the lipid bilayer exerts its effect has remained elusive. Here we focused on ADP ribosylation factor (Arf) GTPases, which orchestrate a variety of regulatory functions in lipid and membrane trafficking, and their activation by the guanine-nucleotide exchange factor (GEF) Brag2, which controls integrin endocytosis and cell adhesion and is impaired in cancer and developmental diseases. Biochemical and structural data are available that showed the exceptional efficiency of Arf activation by Brag2 on membranes. We determined the high-resolution crystal structure of unbound Brag2 containing the GEF (Sec7) and membrane-binding (pleckstrin homology) domains, revealing that it has a constitutively active conformation. We used this structure to analyze the interaction of uncomplexed Brag2 and of the myristoylated Arf1/Brag2 complex with a phosphatidylinositol bisphosphate (PIP₂) -containing lipid bilayer, using coarse-grained molecular dynamics. These simulations revealed that the system forms a close-packed, oriented interaction with the membrane, in which multiple PIP₂ lipids bind the canonical lipid-binding site and unique peripheral sites of the PH domain, the Arf GTPase and, unexpectedly, the Sec7 domain. We cross-validated these predictions by reconstituting the binding and kinetics of Arf and Brag2 in artificial membranes. Our coarse-grained structural model thus suggests that the high efficiency of Brag2 requires interaction with multiple lipids and a well-defined orientation on the membrane, resulting in a local PIP₂ enrichment, which has the potential to signal toward the Arf pathway.

Keywords: crystallography; guanine-nucleotide exchange factor; lipid; molecular dynamics; small GTPase.

Fig. 1.

Crystallographic analysis of uncomplexed Brag2. (A) Structure of unbound Brag2. Regions discussed in the text are indicated. The Sec7 domain is in pink, the linker in yellow, and the PH domain in blue. The same color scheme is used in all figures. (B) Overlay of Brag2 from P2₁2₁2₁ space group (light pink) and C2₁ space group (dark pink), showing the flexibility of the Sec7 C-terminal subdomain. The flexible hinge region is in orange. The orientation is as in A. (C) Electrostatic potential surface of unbound Brag2. The view is rotated by 90° with respect to A.

Fig. S1.

Crystallographic analysis of unbound Brag2^Sec7-PH. (A) Omit map of the K610-L620 loop in the linker region (space group P2₁2₁2₁). (B) Superposition of unbound Brag2 (present work, space group P2₁2₁2₁, light colors) and Arf1-bound Brag2 (dark colors). The rmsd between the two structures is 0.87 Å. The color-coding of the Sec7, linker, and PH domains is as in Fig. 1A. Arf is shown in gray. The orientation is similar to that in Fig. 1A. (C) The membrane-facing surface of the linker and PH domains, showing exposed positively charged residues. The β-strand or the loops between β-strand to which each residue belongs is indicated. The alternative PIP₂-binding site on the outer face of the β1-β2 strands is indicated by an arrow. The view is rotated by 90° with respect to Fig. 1A.

Fig. 2.

Coarse-grained MD simulations of Brag2 on a membrane showing multiple PIP₂ lipid-binding sites. (A) Coarse-grained MD simulation of Brag2 on a membrane bilayer containing PIP₂ lipids at the end of the simulation, for one run. Multiple PIP₂s bind to the protein at various interaction sites at the end of the simulation. The PC lipids and the tails of the PS and PIP₂ lipids are not shown for clarity. (B) Close-up of Brag2 at the end of the simulation run, for one run. The protein binds primarily to PIP₂ molecules in the membrane at multiple binding sites. The view is rotated by 90° with respect to A. (C) Time-averaged number of interactions between Sec7 domain residues and PIP₂ lipids. The Inset shows a close-up of the interactions of the residues in the α8-α9 loop. Additional interactions are also seen in the loop between the Sec7 domain and the linker (residues 580–599). (D) Time-averaged number of interactions between residues of the linker and the PH domain, and PIP₂ lipids. See Fig. 1A and Fig. S1C for the localization of the structural elements bearing these residues. Positively charged residues (lysines and arginines) are in dark blue. PIP₂ lipids are in red; PS lipids are in orange; PC lipids are in gray.

Fig. S2.

Coarse-grained MD simulations of Brag2 on a membrane showing multiple PIP₂ lipid-binding sites. Coarse-grained MD simulation of Brag2 on a membrane bilayer containing PIP₂ lipids at the start (Left) and at the end (Right) of each simulation run. Multiple PIP₂s bind to the protein at various interaction sites at the end of the simulation. The PC lipids and the tails of the PS and PIP₂ lipids are not shown for clarity.

Fig. S3.

The ^myrArf/Brag2 model used for CG-MD simulations. (A) Model of the complex used for the CG-MD simulations. (B) Close-up views of ^myrArf/Brag2 at the end of the simulation run shown in Fig. 3A.

Fig. 3.

Coarse-grained MD simulations of ^myrArf/Brag2 on a membrane showing multiple PIP₂ lipid-binding sites. (A) Close-up of ^myrArf/Brag2 at the end of the simulation run, for one run. Both ^myrArf and Brag2 bind primarily to PIP₂ molecules in the membrane at multiple binding sites. (B) Time-averaged number of interactions between Sec7 domain residues and PIP₂ lipids. (C) Time-averaged number of interactions between the linker and PH domain residues of Brag2 and PIP₂ lipids. (D) Time-averaged number of interactions between Arf residues and PIP₂ lipids. Color-coding for Brag2 and lipids is as in Figs. 1 and 2. Arf is in green.

Fig. S4.

Coarse-grained MD simulations of ^myrArf/Brag2 on a membrane showing multiple PIP₂ lipid-binding sites. Coarse-grained MD simulation of ^myrArf/Brag2 on a membrane containing PIP₂ lipids at the start of the simulation (Left) and at the end (Right) of each simulation run. Multiple PIP₂s bind to the proteins at various interaction sites at the end of the simulations. The PC lipids and the tails of the PS and PIP₂ lipids are not shown for clarity.

Fig. 4.

Interaction of Brag2 with PIP₂ and PIP₂-containing membranes. (A) Flotation analysis of the interaction of Brag2 with liposomes. The His-tagged construct used in this experiment is depicted. Liposomes contained either PIP₂ or NiNTA lipids. B, bottom fraction, containing unbound proteins; T, top fraction, containing liposome-bound proteins. (B) Fluorescence kinetics trace showing the GEF activity of Brag2 (2 nM) toward myristoylated Arf1 (0.4 µM) in the presence of 100 µM PIP₂ liposomes (black) or NiNTA liposomes (gray). The histogram shows the corresponding k_obs values. (C) Analysis of binding of Brag2 to PIP₂-C4 using a thermal shift assay. (D) Liposome flotation analysis of the interaction of Brag2 with liposomes containing increasing PIP₂ concentrations. (E) Fraction of liposome-bound Brag2 as a function of PIP₂ concentration. The Hill plot analysis shown in Fig. S5A indicates that PIP₂ lipids bind to Brag2 in a positively cooperative manner. (F) Analysis of the GEF efficiency on membranes of Brag2 carrying mutations in the Sec7 domain. Liposomes contained either PIP₂ or PS as a source of anionic lipids. The position of the mutations in the Sec7 domain is shown. Composition of liposomes is given in Table S2.

Fig. S5.

Membrane interactions and activity of Brag2. (A) Hill plot analysis of liposome-bound Brag2 as a function of PIP₂ concentration shown in Fig. 4E. This analysis indicates that PIP₂ lipids bind to Brag2 in a positively cooperative manner. (B) Flotation analysis of the interaction of Brag2 with liposomes containing either PIP₂ or PIP₂/PS lipids as a source of anionic lipids. B, bottom fraction containing unbound proteins; T, top fraction, containing liposome-bound proteins. (C) Analysis of the GEF efficiency of Brag2 on liposomes containing either PIP₂ or PIP₂/PS. (D) Comparison of the GEF efficiencies in solution of wild-type Brag2^Sec7-PH and the K549A-R552A mutant.