Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC2238750&req=5

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.

Mentions: In order to provide further evidence for the interaction of Eps15 with the N-terminal EH domain, we performed a pull-down assay using recombinant deletion constructs of Eps15, spanning the DPF region. The GST fusions were incubated with a Hek293 cell extract. Affinity purified proteins were resolved by SDS-PAGE and analyzed by western-blotting using an anti-Eps15 antibody to identify endogenous Eps15. The result (Fig. 6) shows an association that involves DPF triplets at the C-terminal end of Eps15. Although these experiments don't definitively prove a direct DPF/EH interaction in Eps15, as we cannot exclude the involvement of accessory proteins, they confirm a plausible role of EH-DPF interaction in the stabilization of Eps15 tetramers, as suggested by other authors [16].


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

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

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.
Mentions: In order to provide further evidence for the interaction of Eps15 with the N-terminal EH domain, we performed a pull-down assay using recombinant deletion constructs of Eps15, spanning the DPF region. The GST fusions were incubated with a Hek293 cell extract. Affinity purified proteins were resolved by SDS-PAGE and analyzed by western-blotting using an anti-Eps15 antibody to identify endogenous Eps15. The result (Fig. 6) shows an association that involves DPF triplets at the C-terminal end of Eps15. Although these experiments don't definitively prove a direct DPF/EH interaction in Eps15, as we cannot exclude the involvement of accessory proteins, they confirm a plausible role of EH-DPF interaction in the stabilization of Eps15 tetramers, as suggested by other authors [16].

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