title: Molecular basis of phosphatidylinositol 4-phosphate and ARF1 GTPase recognition by the FAPP1 pleckstrin homology (PH) domain.
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.
authors: Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
authors: Ju
authors: J
authors: He
authors: Jordan L
authors: JL
authors: Scott
authors: Annie
authors: A
authors: Heroux
authors: Siddhartha
authors: S
authors: Roy
authors: Marc
authors: M
authors: Lenoir
authors: Michael
authors: M
authors: Overduin
authors: Robert V
authors: RV
authors: Stahelin
authors: Tatiana G
authors: TG
authors: Kutateladze
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D018360:Crystallography, X-Ray; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry* / Q000378:metabolism; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D021601:trans-Golgi Network / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C487037:PLEKHA3 protein, human; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D020823:ADP-Ribosylation Factor 1