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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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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).
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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).

Mentions: In order to confirm the EH targets inferred by the pull down experiment, we synthesized the full-length human Eps15 protein as 15 amino acid long peptides, overlapping by 12 amino acids, using the SPOT synthesis method [21]. The membrane was incubated with the EH domain of POB1 fused to GST and probed with an anti-GST antibody (Fig. 4). The results are partially in agreement with the conclusions drawn from the pull-down experiments. POB1_EH binds to DPF peptides in the regions 623–633 (a) and 647–674 (b); these findings are consistent with the pull down results as the sequences (a) and (b) are also present in the C and D constructs (see Fig. 3A). In addition, two more regions, both containing DPF peptides and encompassing respectively amino acids 592–606 (c) and 796–813 (d) also associate to POB1_EH domain. However, both regions, which may contribute to stabilize binding, are outside the fragments used in the pull-down experiment. Finally, the spots encompassing the sequence of the N construct in the GST pull-down fail to associate with the EH domain.


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)

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

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

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).
Mentions: In order to confirm the EH targets inferred by the pull down experiment, we synthesized the full-length human Eps15 protein as 15 amino acid long peptides, overlapping by 12 amino acids, using the SPOT synthesis method [21]. The membrane was incubated with the EH domain of POB1 fused to GST and probed with an anti-GST antibody (Fig. 4). The results are partially in agreement with the conclusions drawn from the pull-down experiments. POB1_EH binds to DPF peptides in the regions 623–633 (a) and 647–674 (b); these findings are consistent with the pull down results as the sequences (a) and (b) are also present in the C and D constructs (see Fig. 3A). In addition, two more regions, both containing DPF peptides and encompassing respectively amino acids 592–606 (c) and 796–813 (d) also associate to POB1_EH domain. However, both regions, which may contribute to stabilize binding, are outside the fragments used in the pull-down experiment. Finally, the spots encompassing the sequence of the N construct in the GST pull-down fail to associate with the EH domain.

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