Deepti Karandur
Agata Nawrotek
Yvonne Stamp
Agata Stamp


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

Deepti Karandur*, Agata Nawrotek*, John Kuriyan and Jacqueline Cherfils
*these authors contributed equally to the work

Proc. Natl. Acad. Sci. 2017 Sep 18. pii: 201707970. [Epub ahead of print]     (local copy)

Abstract

Lipidated small GTPases and their regulators need to bind to membranes in order 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 is 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 towards the Arf pathway.

Figures from the paper

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Figure 1 from paper

Figure 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.

Figure 2 from paper

Figure 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° degrees with respect to A.


Figure 2 from paper

Figure 2 continued.


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. (lysines and arginines) are in dark blue. PIP2 lipids are in red; PS lipids are in orange; PC lipids are in gray.

Figure 3 from paper

Figure 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. Timeaveraged number of interactions between Sec7 domain residues and PIP2 lipids.
C. Time-averaged number of interactions between the linker and PHdomain 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.

Figure 4 from paper

Figure 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) towards myristoylated Arf1 (0.4 µM) in the presence of 100 µM PIP2 liposomes (black) or NiNTA liposomes (gray). The histogram shows the corresponding kobs 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.

Figure 4 from paper

Figure 4 continued.


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 liposomesis given in Table S2.


Supplemental figures from the paper

(Click on the small image to get a higher-resolution version.)
Supplemental Figure 1 from paper

Supplementary Figure 1 - Crystallographic analysis of unbound Brag2Sec7-PH.


A. Omit map of the K610-L620 loop in the linker region (space group P212121).
B. Superposition of unbound Brag2 (this 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 Figure 1A. Arf is shown in grey. The orientation is similar to that in Figure 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.
Supplemental Figure 2 from paper

Supplementary Figure 2 - 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 the simulation, for 3 runs. 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.

Supplemental Figure 3 from paper

Supplementary Figure 3 - 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.

Supplemental Figure 3 from paper

Supplementary Figure 4 - 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), for 3 runs. 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.

Supplemental Figure 5 from paper

Supplementary Figure 5 - 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 Brag2Sec7-PH and the K549A-R552A mutant.


Supplemental movies from the paper

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Supplemental Figure 1 from paper

Supplementary Movie 1 - CG-MD of unbound Brag2Sec7-PH




Supplemental Figure 2 from paper

Supplementary Movie 2 - CG-MD of myrArf1-bound Brag2Sec7-PH