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. 2011 May 27;286(21):18650-7.
doi: 10.1074/jbc.M111.233015. Epub 2011 Mar 22.

Molecular Basis of Phosphatidylinositol 4-phosphate and ARF1 GTPase Recognition by the FAPP1 Pleckstrin Homology (PH) Domain

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Free PMC article

Molecular Basis of Phosphatidylinositol 4-phosphate and ARF1 GTPase Recognition by the FAPP1 Pleckstrin Homology (PH) Domain

Ju He et al. J Biol Chem. .
Free PMC article

Abstract

Four-phosphate-adaptor protein 1 (FAPP1) regulates secretory transport from the trans-Golgi network (TGN) to the plasma membrane. FAPP1 is recruited to the Golgi through binding of its pleckstrin homology (PH) domain to phosphatidylinositol 4-phosphate (PtdIns(4)P) and a small GTPase ADP-ribosylation factor 1 (ARF1). Despite the critical role of FAPP1 in membrane trafficking, the molecular basis of its dual function remains unclear. Here, we report a 1.9 Å resolution crystal structure of the FAPP1 PH domain and detail the molecular mechanisms of the PtdIns(4)P and ARF1 recognition. The FAPP1 PH domain folds into a seven-stranded β-barrel capped by an α-helix at one edge, whereas the opposite edge is flanked by three loops and the β4 and β7 strands that form a lipid-binding pocket within the β-barrel. The ARF1-binding site is located on the outer side of the β-barrel as determined by NMR resonance perturbation analysis, mutagenesis, and measurements of binding affinities. The two binding sites have little overlap, allowing FAPP1 PH to associate with both ligands simultaneously and independently. Binding to PtdIns(4)P is enhanced in an acidic environment and is required for membrane penetration and tubulation activity of FAPP1, whereas the GTP-bound conformation of the GTPase is necessary for the interaction with ARF1. Together, these findings provide structural and biochemical insight into the multivalent membrane anchoring by the PH domain that may augment affinity and selectivity of FAPP1 toward the TGN membranes enriched in both PtdIns(4)P and GTP-bound ARF1.

Figures

FIGURE 1.
FIGURE 1.
The crystal structure of the PH domain of FAPP1 (C94S) determined at 1.9 Å resolution. a, architecture of FAPP1: the amino-terminal PH domain and a proline-rich motif. b, ribbon diagram of the FAPP1 PH structure.
FIGURE 2.
FIGURE 2.
The PtdIns(4)P-binding site of the FAPP1 PH domain. a, superimposed 1H,15N HSQC spectra of 15N-labeled FAPP1 PH collected during titration with C4-PtdIns(4)P (PI4P) or the lipid head group, Ins(1,4)P2. The spectra are color-coded according to the concentration of the ligands. b, the histogram shows normalized chemical shift changes induced in the backbone amides of the PH domain by PtdIns(4)P. c, residues that display significant chemical shift change in b are labeled on the FAPP1 PH domain surface and colored red, orange, and yellow for large, medium, and small changes, respectively. d, binding affinities of the wild type and mutant FAPP1 PH domain for POPC/POPE/PI4P (75:20:5) vesicles were measured by SPR. e, representative binding isotherms generated from saturation response values at respective FAPP1 PH concentrations were used to calculate Kd. RU, resonance units. Error bars indicate S.D. f, overlay of the β-barrels of the FAPP1 PH domain (red) and the PtdIns(3,4,5)P3-bound GRP1 PH domain (1FGY). PtdIns(3,4,5)P3 is shown as a stick model and colored yellow. Selected hydrogen bonds in the GRP1 complex are depicted as gray lines.
FIGURE 3.
FIGURE 3.
PtdIns(4)P binding is pH-sensitive. a, the specificity of FAPP1 PH was assessed by a protein-lipid overlay assay. S1P, sphingosine 1-phosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,5)P2, phosphatidylinositol 3,5-bisphosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PA, phosphatidic acid; PS, phosphatidylserine; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PI3P, phosphatidylinositol 3-phosphate; PI5P, phosphatidylinositol 5-phosphate; PE, phosphatidylethanolamine; PC, phosphatidylcholine. b, superimposed 1H,15N HSQC spectra of 15N-labeled FAPP1 PH collected during the gradual addition of C4-PtdIns(4)P at pH 6.5 and 7.4. c, binding affinities of the wild type FAPP1 PH domain for POPC/POPE/PI4P (75:20:5) vesicles as measured by SPR. Representative binding isotherm at pH 6.5 was used to calculate Kd values. RU, resonance units. Error bars indicate S.D.
FIGURE 4.
FIGURE 4.
Binding of the FAPP1 PH domain to PtdIns(4)P is necessary for membrane tubulation, pelleting, and insertion. a, top panels, POPC/POPE (80:20) and POPC/POPE/PI4P (75:20:5) membrane sheets labeled with FM 2-10 dye. Middle panels, 2.5 mg/ml FAPP1 PH was injected. Images are shown after 5 min (left) and 2 min (right) of incubation with FAPP1 PH. (bottom panel) POPC/POPE/PI4P membrane sheets after 5 min of incubation with 2.5 mg/ml K45A FAPP1 PH. PC, phosphatidylcholine; PE, phosphatidylethanolamine. b, the insertion of the FAPP1 PH domain into a POPC/POPE (80:20) monolayer (open circles) and a POPC/POPE/PtdIns(4)P (75:20:5) monolayer (filled circles). c, the formation of narrow membrane tubules caused by WT or mutated FAPP1 PH was examined by differential interference contrast microscopy. d, graphs showing normalized pelleted fraction of WT and mutant FAPP1 PH plotted as a function of the initial protein concentration.
FIGURE 5.
FIGURE 5.
The ARF1-binding site of the FAPP1 PH domain. a, superimposed 1H,15N TROSY spectra of 15N-labeled FAPP1 PH collected as unlabeled ARF1 Q71L was titrated in. The spectra are color-coded according to the concentration of ARF1 Q71L. b, the histogram shows normalized chemical shift changes induced in the backbone amides of the PH domain by ARF1 Q71L. c, residues that display significant chemical shift change in b are labeled on the ribbon diagram of FAPP1 PH and colored red and pink for large and medium changes, respectively. d, superimposed 1H,15N TROSY spectra of 15N-labeled ARF1 Q71L recorded while unlabeled FAPP1 PH was added stepwise.
FIGURE 6.
FIGURE 6.
The GTP-bound conformation of ARF1 is essential. a and b, mutations of the ARF1-binding site residues disrupt binding. Pulldown assays and the histogram show that His-ARF1 Q71L is precipitated by wild type or mutant GST-FAPP1 PH bound to the glutathione-Sepharose beads. Error bars indicate S.D. c, superimposed 1H,15N TROSY spectra of 15N-labeled FAPP1 PH collected as wild type ARF1 was titrated in. d, binding affinities of the FAPP1 PH domain for ARF1 as measured by SPR. Kinetic curves were used to calculate Kd and koff.
FIGURE 7.
FIGURE 7.
Molecular mechanism of PtdIns(4)P and ARF1 recognition. a, the residues of FAPP1 PH most perturbed upon interaction with either PtdIns(4)P, ARF1 Q71L, or both ligands are mapped on the surface of the PH domain and colored blue, red, and purple, respectively. b, superimposed 1H,15N HSQC spectra of FAPP1 PH (left) and 1H,15N TROSY spectra of FAPP1 PH prebound to ARF1 Q71L collected during titration with Ins(1,4)P2. c, superimposed 1H,15N TROSY spectra of FAPP1 PH recorded as first C4-PtdIns(4)P and then ARF1 Q71L were titrated in. d, the histogram shows normalized chemical shift changes induced in the backbone amides of the C4-PtdIns(4)P-bound FAPP1 PH domain by ARF1 Q71L. e, a model of anchoring of FAPP1 PH to the Golgi membranes via the double interaction with PtdIns(4)P lipid and myristoylated GTP-bound ARF1.

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