Binding to DPF-motif by the POB1 EH domain is responsible for POB1-Eps15 interaction

Abstract

Background: Eps15 homology (EH) domains are protein interaction modules binding to peptides containing Asn-Pro-Phe (NPF) motifs and mediating critical events during endocytosis and signal transduction. The EH domain of POB1 associates with Eps15, a protein characterized by a striking string of DPF triplets, 15 in human and 13 in mouse Eps15, at the C-terminus and lacking the typical EH-binding NPF motif.

Results: By screening a multivalent nonapeptide phage display library we have demonstrated that the EH domain of POB1 has a different recognition specificity since it binds to both NPF and DPF motifs. The region of mouse Eps15 responsible for the interaction with the EH domain of POB1 maps within a 18 amino acid peptide (residues 623-640) that includes three DPF repeats. Finally, mutational analysis in the EH domain of POB1, revealed that several solvent exposed residues, while distal to the binding pocket, mediate specific recognition of binding partners through both hydrophobic and electrostatic contacts.

Conclusion: In the present study we have analysed the binding specificity of the POB1 EH domain. We show that it differs from other EH domains since it interacts with both NPF- and DPF-containing sequences. These unusual binding properties could be attributed to a different conformation of the binding pocket that allows to accommodate negative charges; moreover, we identified a cluster of solvent exposed Lys residues, which are only found in the EH domain of POB1, and influence binding to both NPF and DPF motifs. The characterization of structures of the DPF ligands described in this study and the POB1 EH domain will clearly determine the involvement of the positive patch and the rationalization of our findings.

Figure 1

Domain organization of human POB1 and human Eps15 proteins. The domain structure of hPOB1 (Swiss Prot entry: Q8NFH8) and of hEps15 (Swiss Prot entry: P42567) and their binding partners are illustrated ([17, 26, 30-36]). Abbreviations: EH, Eps15 Homology; RalBP1-BD, RalA binding protein 1-binding protein; CC, Coiled-Coil region; UIM, Ubiquitin Interacting Motif; Epsin, Eps-15 Interacting protein; Eps-15, Epidermal growth factor receptor pathwat substrate 15; Grb2, growth factor receptor-bound protein 2; PAG2, Paxillin-associated protein with ARFGAP activity 2; AP180, Clathrin coat assembly protein AP180; Hrb, HIV-1 Rev-binding protein.

Figure 2

The EH domain of POB1 binds to both NPF and DPF-containing peptides. Above: phage clones displaying different peptides were adsorbed to a plastic microtiter plate and incubated with the EH domain of POB1 fused to GST and GST alone as negative control. The bound domain was identified with an anti-GST antibody and a secondary antibody linked to alkaline phosphatase; wt is a phage not exposing any ectopic peptide. Below: sequences of the selected peptides are aligned with respect to the NPF and DPF motifs.

Figure 3

The EH domain of POB1 binds to an 18 amino acids fragment comprising residues 623–640 and including three DPF. (A) Schematic representation of the C-terminal DPF-region of mouse Eps15, spanning amino acids 623–750 and comprising 10 DPF tripeptides. The recombinant proteins are progressive COOH-terminal deletions of 10 amino acids fused to the GST. The bars represent the protein fragments that are retained in the recombinant protein. The DPF motifs are indicated by asterisks. (B) Lysates of Hek 293Phoenix expressing Myc-POB1 were incubated with GST or equal amounts of the recombinant proteins bound to glutathione-sepharose beads. Adsorbed proteins were identified with anti-Myc antibody. In the lower panel the GST fusions are visualized with Coomassie staining.

Figure 4

POB1 associates with DPF-containing peptides in the C-terminus of Eps15. (A) Full-length human Eps15 was synthesized as 15 amino acid peptides, overlapping by 12 amino acids, on a cellulose membrane using the SPOT synthesis method [21]. The membrane was incubated with the EH domain of POB1 fused to the GST and probed with an anti-GST antibody. The groups of spots considered to be positives are indicated as a, b, c and d. Positives controls, binding to secondary antibodies, are indicated with rectangles. (B) Amino acid sequence of human Eps15. The N-terminal region comprising the three EH domains is underlined, sequences corresponding to the spots considered to be positives are outlined. Residues corresponding to the indicated regions are: a (aa 623–633), b (aa 647–674), c (aa 592–606) and d (aa 796–813).

Figure 5

The amino-terminal EH domain of Eps15 associates with full length Eps15. (A) The EH domain of POB1 and the N-terminal region of Eps15 comprising three EH domains (EH1-EH2-EH3) were tested in a pull-down experiment from Hek293 to evaluate the relative binding efficiency towards endogenous Eps15. (B) The isolated GST-EH domains (EH1, EH2, EH3) and the N-terminal region of Eps15 were tested in a similar experiment. The input lane corresponds to 0,1% of the lysate. Relative binding efficiencies represent the quantification of the Western blotting using the Image Quant Software. In the lower panel, the GST fusions are visualized by Coomassie staining.

Figure 6

The DPF region of Eps15 binds to Eps15 in a pull-down experiment. 50 μg of the Eps15 recombinant proteins spanning amino acids 623–750 and comprising 10 DPF tripeptides, as described in Figure 3, were incubated with a cell extract from Hek293. Bound proteins were resolved by SDS-PAGE and analyzed by western-blotting using an anti-Eps15 antibody.

Figure 7

Structural analysis of the NPF-binding pocket of Eps15 (EH2) and POB1 EH domain. Molecular surface representations of the NPF-binding pocket of the Eps15 EH2, PDB code: 1EH2 (A), Eps15 EH1, PDB code: 1QJT (B) and POB1, PDB code: 1IQ3 (C) EH domains. Residues in the hydrophobic groove are coloured in red, residues which line the edge of the binding pocket are in green while the gate charged residues are in blue. (B) Left panel: representation of the classical binding pocket of Eps15 EH1 domain. Right panel: residues distant from the binding pocket, which have been discussed in the text, are mapped on the molecular surface of Eps15. Lys 21 indicates the position of the binding pocket. (C) Left panel: representation of the classical binding pocket of POB1 EH domain. Right panel: residues mutated in this study are mapped on the molecular surface of POB1. Lys 307 indicates the position of the binding pocket. Lysine residues are coloured in blue, Phe344 and Ile347 in red and Gln289 in orange. Molecular surfaces were generated with PyMol.

Figure 8

Mutations in lysine residues distant from the binding pocket affect the interaction of POB1 with Eps15. Residues K312, W324, F344 and K351 in the POB1-EH domain were changed into Ala and the recombinant proteins were tested in an Eps15 pull-down assay from Hek293. Bound proteins were resolved by SDS-PAGE and analyzed by western-blotting using an anti-Eps15 antibody. In the lower panel, the GST fusions are visualized by Coomassie staining. Relative binding efficiencies represent the quantification of the Western blotting using the Image Quant Software.