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

Santonico E, Panni S, Falconi M, Castagnoli L, Cesareni G - BMC Biochem. (2007)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Rome Tor Vergata, Rome, Italy. Elena.Santonico@uniroma2.it

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.

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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.
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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.

Mentions: The results so far indicate that the POB1 EH domain can bind DPF peptides displayed on filamentous phage and suggest that this novel binding specificity could be responsible for the recognition of the Eps15 C-terminal domain. To map the Eps15 sequences responsible for binding to POB1 we expressed as GST fusions progressive COOH-terminal (10 amino acids) deletions of the region spanning amino acids 623–743 of mouse Eps15 (Fig. 3A) [18]. Agarose-immobilized GST-Eps15 fusion proteins were tested in a pull-down assay carried out on 293Phoenix cells over-expressing full-length POB1 fused to the Myc epitope. The results reported in Figure 3B indicate that the binding of the POB1 EH domain to the C-terminal region of Eps15 is influenced in a complex manner by the length of the region. Shortening of the 121 amino acid DPF rich fragment initially results in negative modulation of binding (see constructs D, F and G); further shortening, however, restores efficient binding. A C-terminal fragment (N in Fig 3A) is also capable of binding. These results are compatible with the presence of at least two different binding sites with fragment length affecting conformation and, as a consequence, availability of DPF motifs for EH binding.


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

Santonico E, Panni S, Falconi M, Castagnoli L, Cesareni G - BMC Biochem. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2238750&req=5

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.
Mentions: The results so far indicate that the POB1 EH domain can bind DPF peptides displayed on filamentous phage and suggest that this novel binding specificity could be responsible for the recognition of the Eps15 C-terminal domain. To map the Eps15 sequences responsible for binding to POB1 we expressed as GST fusions progressive COOH-terminal (10 amino acids) deletions of the region spanning amino acids 623–743 of mouse Eps15 (Fig. 3A) [18]. Agarose-immobilized GST-Eps15 fusion proteins were tested in a pull-down assay carried out on 293Phoenix cells over-expressing full-length POB1 fused to the Myc epitope. The results reported in Figure 3B indicate that the binding of the POB1 EH domain to the C-terminal region of Eps15 is influenced in a complex manner by the length of the region. Shortening of the 121 amino acid DPF rich fragment initially results in negative modulation of binding (see constructs D, F and G); further shortening, however, restores efficient binding. A C-terminal fragment (N in Fig 3A) is also capable of binding. These results are compatible with the presence of at least two different binding sites with fragment length affecting conformation and, as a consequence, availability of DPF motifs for EH binding.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Rome Tor Vergata, Rome, Italy. Elena.Santonico@uniroma2.it

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.

Show MeSH
Related in: MedlinePlus