Two WXXF-based motifs in NECAPs define the specificity of accessory protein binding to AP-1 and AP-2

Abstract

The adaptor proteins AP-2 and AP-1/GGAs are essential components of clathrin coats at the plasma membrane and trans-Golgi network, respectively. The adaptors recruit accessory proteins to clathrin-coated pits, which is dependent on the adaptor ear domains engaging short peptide motifs in the accessory proteins. Here, we perform an extensive mutational analysis of a novel WXXF-based motif that functions to mediate the binding of an array of accessory proteins to the alpha-adaptin ear domain of AP-2. Using nuclear magnetic resonance and mutational studies, we identified WXXF-based motifs as major ligands for a site on the alpha-ear previously shown to bind the DPW-bearing proteins epsin 1/2. We also defined the determinants that allow for specific binding of the alpha-ear motif to AP-2 as compared to those that allow a highly related WXXF-based motif to bind to the ear domains of AP-1/GGAs. Intriguingly, placement of acidic residues around the WXXF cores is critical for binding specificity. These studies provide a structural basis for the specific recruitment of accessory proteins to appropriate sites of clathrin-coated vesicle formation.

Figure 1

Definition of the α-ear-binding motif in NECAPs. (A) Sequence alignment of the C-terminal most six amino acids of murine NECAP 1 and 2 with NECAP isoforms from Xenopus laevis (Xenopus, gi32484237) and Drosophila melanogaster (Drosophila, gi24642653). The conserved core of the α-ear-binding motif is shaded black. The numbers before and after the sequences indicate amino-acid positions within the protein. The numbering underneath designates positions within the motif. The asterisks indicate free main chain carboxyl groups. (B–D) GST or peptides fused to GST as indicated were precoupled to glutathione–Sepharose and incubated with soluble rat brain extracts. Affinity-selected AP-2 (α-adaptin) was detected by Western blot. An aliquot of brain homogenate (starting material, SM) equal to one-tenth of that added to the fusion proteins was analyzed in parallel. The tetrapeptide cores of the motifs are underlined and bold amino acids indicate the substitutions introduced into the wild-type sequence. (B) Alanine screen for positions −1 and −2 in NECAP 1 and 2. Mutational analysis for W at position 0 (C) and F at position +3 (D). (E) Immunoprecipitation of overexpressed Flag-tagged NECAP 1 variants (Flag) and co-immunoprecipitated AP-2 (α-adaptin) were detected by Western blot. Aliquots of the cell lysates (starting material, SM) equal to one-tenth of that used for the immunoprecipitations were analyzed in parallel.

Figure 2

Definition of the inner core positions. (A–E) GST or peptides fused to GST as indicated were precoupled to glutathione–Sepharose and incubated with soluble rat brain extracts. Affinity-selected AP-2 (α-adaptin) was detected by Western blot. An aliquot of brain homogenate (starting material, SM) equal to one-tenth of that added to the fusion proteins was analyzed in parallel. The tetrapeptide cores of the motifs are underlined and bold amino acids indicate the substitutions introduced into the wild-type sequence. (A/D) Mutational analysis for V at position +1 and for Q at position +2 (B/E). (C) Sequence alignment of potential NECAP-like α-ear-binding motifs in proteins connected to vesicle trafficking. The conserved hydrophobic amino acids at the 0 and +3 positions are shaded black and similar residues are shaded gray. Small letters in superscript indicate distinct copies of the motif within the same protein. AAK1, adaptor-associated kinase 1; BIKe, BMP-2 inducible kinase; GAK, cyclin G-associated kinase; PACSIN, PKC and CK2 substrate in neurons; REPS1, RalBP1-associated Eps domain containing protein 1.

Figure 3

Contribution of downstream negative charge to AP-2 interaction. (A–D) GST or peptides fused to GST as indicated were precoupled to glutathione–Sepharose and incubated with soluble rat brain extracts. Affinity-selected AP-2 (α-adaptin) was detected by Western blot. An aliquot of brain homogenate (starting material, SM) equal to one-tenth of that added to the fusion proteins was analyzed in parallel. The tetrapeptide cores of the motifs are underlined and bold amino acids indicate the substitutions introduced into the wild-type sequence. (A) Contribution of the free carboxyl group in NECAP 1 to AP-2 binding. (B–D) Contribution of the downstream acidic amino acids to AP-2 binding for various motifs in stonin 2 as indicated.

Figure 4

Identification of the α-ear-binding site for WXXF-based motifs. (A) Comparison of ¹H-¹⁵N HSQC spectra of the ¹⁵N-labeled α-ear in the absence (black) or presence (red) of the NECAP 1 peptide (1:1 complex). (B) Magnitude of the amide chemical shift changes ({(Δ¹H shift)²+(Δ¹⁵N shift × 0.2)²}^1/2 in ppm) of the α-ear upon binding CSDPWGSDPWG (epsin, upper panel), CSNWVQFEDDTP (stonin 2, middle panel), and CQAPQPSNWVQF (NECAP 1, lower panel) peptides. The indicated residue numbers correspond to mouse α-adaptin. (C) Amino-acid sequence of the α-ear shown with β-strands in the sandwich domain depicted as green arrows and β-strands and α-helices of the platform domain depicted as blue arrows and rods, respectively. Green shading represents the size of the amide chemical shift changes upon binding of the NECAP1 peptide (dark green: Δδ>0.2; light green: 0.2>Δδ>0.1 ppm). Blue shading represents the size of the amide chemical shift changes upon binding of the epsin peptide (dark blue: Δδ>0.2; light blue: 0.2>Δδ>0.1 ppm). Asterisks mark residues (N713A, Q723A, K727, F740A, and Q782) that, when mutated, affect binding to NECAP 1.

Figure 5

Model of the WVQF core bound to the α-ear. (A) HADDOCK modeled structure of WVQF/α-ear complex. The backbone trace of the α-ear is colored according to the size of the amide chemical shift changes upon binding of the NECAP 1 peptide. (B) Electrostatic potential surface analysis for the WVQF/α-ear complex. The surfaces are color coded, with red indicating negative electrostatic potential (E and D residues) and blue indicating positive potential (K, R, and H residues). (C) GST or GST fusion proteins of wild-type α-ear along with α-ear mutants as indicated were precoupled to glutathione–Sepharose and incubated with soluble rat brain extracts. Affinity-selected Eps15, epsin, amphiphysin I and II, and NECAP 1 were detected by Western blot. An aliquot of brain homogenate (starting material, SM) equal to one-tenth of that added to the fusion proteins was analyzed in parallel.

Figure 6

Determinants establishing binding specificity for WXXF-based α-ear- and GAE-binding motifs. (A) Sequence alignment of the C-terminal region of murine NECAP 1 and 2. Conserved amino acids are shaded black, similar residues are shaded gray. The GAE domain- and α-ear-binding motifs are underlined and arrows indicate the specific interaction of these motifs with different adaptor proteins. The numbers after the sequences indicate amino-acid position within the protein. (B/C) GST or peptides fused to GST as indicated were precoupled to glutathione–Sepharose and incubated with soluble rat brain extracts. Affinity-selected AP-2 (α-adaptin) and AP-1 (γ-adaptin) were detected by Western blot. An aliquot of brain homogenate (starting material, SM) equal to one-tenth of that added to the fusion proteins was analyzed in parallel. The tetrapeptide cores of the motifs are underlined and bold amino acids indicate the substitutions introduced into the wild-type sequence. (B) Step-by-step conversion of the α-ear-binding motif into a GAE-binding motif. (C) Step-by-step conversion of the GAE-binding motif into a α-ear-binding motif. (D) Schematic overview on parameters regulating binding specificity of W–X–X–[FW]-based motifs for interaction with GAE domains (AP-1), AP-2, and the clathrin terminal domain (clathrin). The question mark indicates undefined parameters. (E) GST-NECAP 1 was precoupled to glutathione–Sepharose and for each pull-down 100 pmol were incubated with 0.5 mg soluble brain extract without (no peptide) or in the presence of increasing concentrations of various peptides as indicated: AP-1 wild-type peptide, CSNDLWGDFSTAS; AP-1 mutant peptide, CSNNLWVDFSTAS; AP-2 wild-type peptide, CQAPQPSNWVQF; AP-2 mutant peptide, CQAPQPSNWGQFAAA. The molar ratio of peptide to fusion protein is indicated and affinity-selected AP-1 (γ-adaptin) and AP-2 (α-adaptin) were detected by Western blot. Aliquots of brain homogenate (starting material, SM) equal to one-tenth of that added to the fusion proteins were analyzed in parallel. (F) COS-7 cells were transfected with the empty expression vector (vector control) or with Flag-tagged NECAP 1 BC variants as indicated and the percentage of cells that fail to endocytose Cy3-labeled transferrin was quantified (mean±s.e.m., n=5).