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. 2014 Mar 4;22(3):421-30.
doi: 10.1016/j.str.2013.12.011. Epub 2014 Jan 23.

Interaction of Fapp1 With Arf1 and PI4P at a Membrane Surface: An Example of Coincidence Detection

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Interaction of Fapp1 With Arf1 and PI4P at a Membrane Surface: An Example of Coincidence Detection

Yizhou Liu et al. Structure. .
Free PMC article

Abstract

Interactions among ADP-ribosylation factors (ARFs), various adaptor proteins, and membrane lipids are essential for intracellular vesicle transport of a variety of cellular materials. Here, we present nuclear magnetic resonance (NMR)-based information on the nature of the interaction of yeast Arf1 (yArf1) and the pleckstrin homology (PH) domain of four-phosphate-adaptor protein 1 (Fapp1) as it occurs at a model membrane surface. Interactions favor a model in which Fapp1 is partially embedded in the membrane and interacts with a membrane-associated Arf1 molecule primarily through contacts between residues in switch I of Arf1 and regions near and under the solution exposed C-terminal extension of the PH domain. The Arf1 binding site on Fapp1-PH is distinct from a positively charged phosphatidylinositol-4-phosphate (PI4P) binding site. A structural model is constructed that supports coincidence detection of both activated ARF and PI4P as a mechanism facilitating Fapp1 recruitment to membranes.

Conflict of interest statement

The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1
FAPP1-PH interacts with PI4P. A, Superimposed TROSY spectra of FAPP1-PH in the presence of 10% DMPC/DHPC (q=0.25) bicelle (red) and bicelle doped with 8mM PI4P (blue). Assignments are indicated for residues that undergo combined chemical shift changes greater than 0.15ppm. B, 31P spectra from serial titration of FAPP1-PH (0–3.5 mM) into 10% DMPC/DHPC (q=0.25) bicelle doped with 2mM PI4P, highlighting the migration and broadening/re-narrowing of the PI4P phosphate monoester signal.
Figure 2
Figure 2
The PI4P binding interface of FAPP1-PH identified through chemical shift perturbations. A, A surface view of FAPP1-PH colored in red for residues of strong chemical shift perturbations (>0.4 ppm) upon adding PI4P and in yellow for those of weak perturbations (<0.4 and >0.2 ppm). B. The electrostatic surface of FAPP1-PH orientated in the same perspective as in A, highlighting the positively-charged pocket for PI4P binding. Positive potentials are shown in blue.
Figure 3
Figure 3
The ARF1 binding interface of FAPP1-PH identified through chemical shift perturbations. A, Residue specific chemical shift perturbations in TROSY spectra of FAPP1-PH in the presence of myr-yARF1•GTPγS in 10% DMPC/DHPC (q=0.25), 0.5% PI4P bicelles. Resonances that disappear have been set to 0.05 ppm. B, The ARF1 binding surface of FAPP1-PH colored in red for residues showing strong chemical shift perturbations upon adding ARF1 and in yellow for those showing weak perturbations. FAPP1-PH is rotated approximately 180° relative to the depiction in Figure 2. Residue numbers are indicated for those discussed in the text.
Figure 4
Figure 4
The FAPP1-PH binding interface of yARF1•GTPγS identified through chemical shift perturbations. A, Residue specific chemical shift perturbations in TROSY spectra of myr-yARF1•GTPγS in 10% DMPC/DHPC (q=0.25), 0.5% PI4P bicelles in the presence of 1.5mM FAPP1-PH. The bars marked with * are histidine residues whose shifts responded to small differences in pH (truncated to 0.05). B, The FAPP1-PH binding surface of ARF1 colored in red for residues of strong chemical shift perturbation and in orange for those of weak perturbations. Residue numbers are indicated for those discussed in the text.
Figure 5
Figure 5
The ARF1 binding interface of FAPP1-PH identified through paramagnetic relaxation enhancements. A, Superimposed fast HSQC spectra of 0.3mM 2D-15N FAPP1-PH mixed with 0.9mM unlabeled oxidized yARF1.GTPγS -T55C-MTSSL (blue) and reduced yARF1.GDP-T55C-MTSSL (red). B, The binding surface of FAPP1-PH colored in red for residues undergoing strong PRE effects and in orange for those undergoing weaker PRE effects. PRE effects reflect spatial proximity to the spin-labeled site (T55) of ARF1. Residue numbers are indicated for those discussed in the text.
Figure 6
Figure 6
The FAPP1-PH/yARF1 complex modeled by HADDOCK using chemical shift perturbation and PRE data as the experimental constraints. FAPP1-PH is colored in cyan with residues of strong and weak chemical shift perturbations upon adding yARF1•GTPγS and used in the calculations shown in purple and magenta respectively; residues strongly affected by PREs and used in the calculations are shown in blue. yARF1•GTPγS is colored in green with residues of strong and weak chemical shift perturbations upon adding FAPP1-PH shown in red and orange respectively.
Figure 7
Figure 7
The FAPP1-PH/PI4P complex modeled by HADDOCK using chemical shift perturbation data as the experimental constraints. Residues of significant chemical shift perturbations upon adding PI4P and used in the calculation are shown in magenta. Residue numbers are indicated for those discussed in the text.
Figure 8
Figure 8
A model of FAPP1-PH/PI4P/ARF1 ternary complex on a small bicelle surface. The HADDOCK models for the FAPP1-PH/yARF1 complex and the FAPP1-PH/PI4P complex are superimposed through the common partner FAPP1-PH, to obtain a model for the ternary complex. This complex is further superimposed through ARF1 structures with our previously published ARF1/bicelle model, to obtain the FAPP1-PH/PI4P/ARF1 ternary complex on the small bicelle surface.

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