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


CONCLUSIONS
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.
D033965:Adaptor Protein Complex alpha Subunits / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000235:genetics; D016384:Consensus Sequence; D004705:Endocytosis; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D010750:Phosphoproteins / Q000235:genetics; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D033965:Adaptor Protein Complex alpha Subunits; D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C486206:EPS15 protein, human; C082729:Eps15 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D010750:Phosphoproteins; C489722:REPS2 protein, human; D011994:Recombinant Proteins