title: The NECAP PHear domain increases clathrin accessory protein binding potential.
abstract: AP-2 is a key regulator of the endocytic protein machinery driving clathrin-coated vesicle (CCV) formation. One critical function, mediated primarily by the AP-2 alpha-ear, is the recruitment of accessory proteins. NECAPs are alpha-ear-binding proteins that enrich on CCVs. Here, we have solved the structure of the conserved N-terminal region of NECAP 1, revealing a unique module in the pleckstrin homology (PH) domain superfamily, which we named the PHear domain. The PHear domain binds accessory proteins bearing FxDxF motifs, which were previously thought to bind exclusively to the AP-2 alpha-ear. Structural analysis of the PHear domain reveals the molecular surface for FxDxF motif binding, which was confirmed by site-directed mutagenesis. The reciprocal analysis of the FxDxF motif in amphiphysin I identified distinct binding requirements for binding to the alpha-ear and PHear domain. We show that NECAP knockdown compromises transferrin uptake and establish a functional role for NECAPs in clathrin-mediated endocytosis. Our data uncover a striking convergence of two evolutionarily and structurally distinct modules to recognize a common peptide motif and promote efficient endocytosis.
authors.affiliation: Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Ritter
authors.forename: Alexei Yu
authors.initials: AY
authors.lastname: Denisov
authors.forename: Jacynthe
authors.initials: J
authors.lastname: Philie
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Allaire
authors.forename: Valerie
authors.initials: V
authors.lastname: Legendre-Guillemin
authors.forename: Peter
authors.initials: P
authors.lastname: Zylbergold
authors.forename: Kalle
authors.initials: K
authors.lastname: Gehring
authors.forename: Peter S
authors.initials: PS
authors.lastname: McPherson
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D002966:Clathrin; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C075637:amphiphysin