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.

Figure 1

The NECAP fold. (A) Schematic representation of the organization of NECAP 1. The evolutionarily conserved N-terminal region (amino acids 1–178) is represented in green and the portion encoding the PHear fold (amino acids 1–133) is indicated by the purple line. The C-terminal binding motifs for AP-1/GGAs (blue) and AP-2 (brown) are assigned to the corresponding adaptor protein. (B) Ribbon representation of the solution structure of the NECAP 1 PHear domain with β-strands and α-helices indicated. (C) Stereoview of the backbone superposition of 10 low-energy structures.

Figure 2

NECAPs interact with amphiphysins. (A, B) GST fusion proteins encoding the NECAP 1 PHear domain, NECAP 1 aa 1–178, NECAP 1 aa 129–178, NECAP 2 aa 1–177, or GST alone pre-coupled to glutathione–Sepharose were incubated with Triton X-100-solubilized rat brain extract or purified amphiphysin I as indicated, and amphiphysin binding was analyzed by Western blot. (C) Co-immunoprecipitation of endogenous NECAP 1 and amphiphysins from Triton X-100-solubilized rat brain extract using antibodies directed against amphiphysin (1874, which recognizes both amphiphysin I and II) or NECAP 1. Immunoprecipitated proteins were detected by Western blot. For all binding studies, 10% of the protein amount used in each reaction is analyzed as starting material (SM).

Figure 3

Identification of the NECAP 1-binding region in amphiphysin I. (A) Domain structure of amphiphysin I indicating the N-terminal BAR domain followed by the neuronal insert domain, a linker region, and the C-terminal SH3 domain, and schematic presentation of N- and C-terminal deletion constructs. Amino-acid positions in front and behind the fragment indicate the protein borders. (B) Pull-down experiments with Triton X-100-solubilized rat brain extract using GST fusion proteins of the amphiphysin I variants indicated in panel A or GST alone pre-coupled to glutathione–Sepharose. Binding of clathrin and NECAP 1 was analyzed by Western blot. (C) Schematic presentation of binding motifs for endophilin A1 (PxxP), clathrin (LLDLD, PWDLW), and AP-2 (FxDxF, DPF) within the neuronal insert domain of amphiphysin I, and overview of N- and C-terminal deletion constructs with amino-acid positions indicating the protein borders. (D) Pull-down experiments with Triton X-100-solubilized rat brain extract using GST fusion proteins of the amphiphysin I variants indicated in panel C or GST alone pre-coupled to glutathione–Sepharose. Binding of clathrin, AP-2, and NECAP 1 was analyzed by Western blot. The faint signal for NECAP 1 with the GST fusion protein encoding amino acids 291–380 of amphiphysin I is due to co-migration of NECAP 1 with the fusion protein. (E) Aliquots (100 pmole) of the NECAP 1 PHear domain fused to GST or GST alone pre-coupled to glutathione–Sepharose were incubated with Triton X-100-solubilized rat brain extract in the absence or molar excess of a synthetic peptide encoding the amphiphysin I FxDxF motif (CSFFEDNFPE) as indicated. Binding of amphiphysin I and II was analyzed by Western blot. For all binding studies, 10% of the protein amount used in each reaction is analyzed as starting material (SM).

Figure 4

The FXDXF motif in amphiphysin I mediates interaction with NECAP 1. (A, B) Mapping of amphiphysin residues involved in binding to NECAP 1. (A) ¹⁵N–¹H HSQC spectrum of the ¹⁵N-labeled 39-amino-acid amphiphysin I peptide (amino acids 291–329) with signal assignments. (B) Magnitude of the amide chemical shift changes of the ¹⁵N-labeled 39-amino-acid amphiphysin peptide upon binding of the unlabeled NECAP 1 (the changes for N-terminal residues 291–311 residues are not presented, as their Δδ is less than 0.03 p.p.m.). (C) Pull-down experiments using purified His-tagged α-ear and His-tagged NECAP 1 and GST fusion proteins of amphiphysin I variants as indicated, or GST alone pre-coupled to glutathione–Sepharose. Binding of α-ear and NECAP 1 was analyzed by Western blot. An aliquot equal to 10% of the protein amount used in each reaction is analyzed as starting material (SM). Longer exposures reveal His-α-ear in the SM (data not shown). Aliquots of 30% of the amounts used for each GST fusion protein and GST alone were analyzed in parallel by Coomassie staining to control for protein amounts used in the pull-down experiments.

Figure 5

Identification of the NECAP 1 binding site for FxDxF motifs. (A) Comparison of ¹⁵N–¹H HSQC spectra of ¹⁵N-labeled NECAP 1 in the absence (black) or presence (red) of a synthetic amphiphysin I peptide (CSFFEDNFPE) (at a 2:1 peptide–protein ratio). (B) Magnitude of the amide chemical shift changes of the NECAP 1 residues upon binding of the amphiphysin I insert domain (amino acids 291–445) or the synthetic peptide. (C) Sequence of NECAP 1 with positions of β-strands and α-helices indicated. Color shading represents the size of the amide chemical shift changes (red, Δδ>0.2 and yellow, 0.2>Δδ >0.1 p.p.m.) upon binding of the amphiphysin I insert domain. Asterisks mark residues that when mutated affect binding. (D) Backbone trace of NECAP 1 colored according to the size of the chemical shift changes indicated in panel C. (E) GST fusion proteins encoding wild-type NECAP 1 PHear domain and point mutations as indicated, or GST alone pre-coupled to glutathione–Sepharose were incubated with Triton X-100-solubilized rat brain extract and interaction with amphiphysin I and II was analyzed by Western blot. A total of 10% of the protein amount used in each reaction is analyzed as starting material (SM).

Figure 6

The PHear domain is a general FxDxF motif-binding site implicated in the regulation of clathrin-mediated endocytosis. (A) Pull-down experiments with Triton X-100-solubilized rat brain extract using GST fusion proteins of the α-ear and NECAP variants as indicated, or GST alone pre-coupled to glutathione–Sepharose. Binding of various endocytic accessory proteins was analyzed by Western blot and the motifs involved in α-ear and PHear domain binding are indicated. For all binding studies, 10% of the protein amount used in each reaction is analyzed as starting material (SM). (B) Equal protein amounts of Triton-X100-solubilized COS-7 cells transduced with various combinations of shRNA constructs for simultaneous knockdown of NECAP 1 and 2 or with control shRNA constructs (control D/Q) were analyzed by Western blot for expression levels of various proteins as indicated. The arrowhead indicates the NECAP 2 signal, the band underneath represents a cross-reaction of the antibody. (C) Immunofluorescence analysis for uptake of fluorescently labeled transferrin (Tfn) in COS-7 cells transduced with various combinations of shRNA constructs for simultaneous knockdown of NECAP 1 and 2 or with control shRNA constructs (control D/Q). Cells were allowed to endocytose for 2.5, 5, or 10 min, as indicated. Transduced cells express GFP from an independent expression cassette. (D) Quantification of the transferrin uptake assay described in panel C. The number of independent experiments (No. of indep. exp.), total number of microscopic fields analyzed (total no. of fields), and total number of cells analyzed (total # of cells) are indicated for each time-point and knockdown condition underneath the corresponding bar. ^*P<0.05, ^**P<0.01, and ^***P<0.001.

Figure 7

Comparison of the molecular surfaces of the PHear domain and AP-2 α-ear. (A, B) Presentation of the FxDxF motif-binding surface for the NECAP PHear domain (left) and the α-ear of AP-2 (right). The orientation of the NECAP PHear domain is corresponding to Figure 5D. (A) The surface of both proteins is color-coded, with red indicating negative electrostatic potential and blue indicating positive potential. The amphiphysin I SFFEDNFVP peptide is shown in green. The atomic coordinates for the α-ear/FxDxF motif complex were taken from PDB entry 1KY7. (B) Color-coding (green) highlights amino acids implicated in FxDxF motif binding by NMR for the PHear domain, or shown to contact the motif by co-crystallization for the α-ear. Mutational analysis of amino acids labeled in orange verified their contribution to FxDxF motif binding (this study and Brett et al, 2002).