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. 2009 Dec;18(12):2471-9.
doi: 10.1002/pro.258.

Structural insight into the interaction of proteins containing NPF, DPF, and GPF motifs with the C-terminal EH-domain of EHD1

Fabien Kieken  1 , Marko Jović, Marco Tonelli, Naava Naslavsky, Steve Caplan, Paul L Sorgen
Affiliations

Structural insight into the interaction of proteins containing NPF, DPF, and GPF motifs with the C-terminal EH-domain of EHD1

Fabien Kieken et al. Protein Sci. 2009 Dec.

Abstract

Eps15 homology (EH)-domain containing proteins are regulators of endocytic membrane trafficking. EH-domain binding to proteins containing the tripeptide NPF has been well characterized, but recent studies have shown that EH-domains are also able to interact with ligands containing DPF or GPF motifs. We demonstrate that the three motifs interact in a similar way with the EH-domain of EHD1, with the NPF motif having the highest affinity due to the presence of an intermolecular hydrogen bond. The weaker affinity for the DPF and GPF motifs suggests that if complex formation occurs in vivo, they may require high ligand concentrations, the presence of successive motifs and/or specific flanking residues.

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Figures

Figure 1
Figure 1
Interaction between the EHD1 EH-domain (residues D436-E534) and ligands containing NPF, DPF, and GPF motifs. A) The 15N-HSQC of the EH-domain alone (black) has been overlaid with the EH-domain in complex with 2 mM NPF (red), 3 mM DPF (green), and 4 mM GPF (blue) containing peptides. The resonance peaks, which exhibited the greatest change in chemical shift due to the ligands have been highlighted by circles. The residues colored red were used to calculate the binding affinities.
Figure 2
Figure 2
Binding analysis of the interaction between the EHD1 EH-domain (residues D436-E534) and ligands containing (A) NPF, (B) DPF, and (C) GPF motifs. The dissociation constants (KD) were estimated by non-linear best fitting the chemical shift variation (in ppm) versus the concentration of peptide (KD - NPF: 245 μM ± 35, DPF: 1.2 mM ± 0.1, GPF 2.4 mM ± 0.1). Chemical shift variation was calculated according to the formula Δσ = v((ΔδHN)2 + (ΔδN/5)2. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
Figure 3
Figure 3
Structure of EHD-1 EH-domain in complex with XPF peptides. A-C) Stereoview of the 10 lowest energy structures of the EHD-1 EH-domain in complex with XPF peptides. The structures of the EH-domain in complex with (A) NPF, (B) DPF, and (C) GPF peptides have been superpositioned according to the backbone atoms of the XPF residues (X = N, D, or G). The EH-domain residues involved in the interaction with the XPF residues are Gly464, Ala467, Lys468, Me471, Asn478 Leu481, Gly481, Trp485. The side chains of these EH-domain residues have been labeled (red). (D–F) Close up view of the binding site between the EHD1 EH-domain and XPF peptides. The EH-domain is colored blue and the (D) NPF, (E) DPF, and (F) GPF peptides are colored orange. Residues involved in the direct interaction have been labeled in each panel.
Figure 4
Figure 4
Demonstration that the EHD1 EH-domain is capable of binding to its own GPF motif. Overlay of 15N-HSQCs of the EH-domain (residues E401-E534) collected at different protein concentrations (black: 100 μM; red: 500 μM; and green: 1 mM).
Figure 5
Figure 5
Binding analysis of the interaction between the EHD1 EH-domain (residues D436-E534) and a ligand containing the native EHD1 GPF motif. (A) 15N-HSQC of the EH-domain (residues 436–534) with different concentrations of GPF wild type (EHD1 residues F414–G425) (black: 0 mM; red: 1 mM; green: 2.5 mM; and blue: 5 mM). Resonances used to calculate the dissociation constant (KD) have been circled. The KD was estimated to be 2.3 mM ± 0.3 by non-linear best fitting the chemical shift variation (in ppm) versus the concentration of peptide.
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