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Eps15: A high affinity EH domain complex

Eps15 homology (EH) domains are important mediators of protein-protein interactions in various trafficking processes in the cell. EH domains were shown to bind motifs containing the amino acid residues Asn-Pro-Phe (NPF) in unfolded regions of their binding partners (de Beer et al., 1998; Confalonieri and Di Fiore, 2002; Morgan et al., 2003). So far, interactions between EH-domains and their respective ligands were reported to be of low affinity and also low specificity (Salcini et al., 1997).

We discovered a highly specific, high affinity interaction between Eps15, an essential accessory protein during clathrin mediated endocytosis, and its binding partner Stonin2 (see Rumpf et al., 2008). Stonin2 was shown to act as a specific sorting adaptor for the internalization of synaptotagmin and thus to be a regulator of synaptic vesicle recycling (Diril et al., 2006).

Eps15 is a multi-modular protein that comprises 3 EH domains, a coiled-coil region and a low complexity region at its c-terminus that harbors several peptide motifs, shown to interact with other endocytic proteins, for example the AP-2 complex (Figure 1). Stonin2 consists of a ? homology domain, a Stonin homology (SH) domain and a low complexity N-terminus with 2 NPF motifs.
Domain structure of Eps15 and stonin2

Figure 1: Domain structure of Eps15 and Stonin2

Eps15-EH1-3 binds Stonin2 NPF-region with an apparent affinity of 0.15 uM, about three orders of magnitude tighter than observed for other EH-NPF complexes.

This high-affinity interaction is mediated by the second EH domain of Eps15 only (Figure 2). EH1 binds with low affinity to the Stonin2 NPF region, while we cannot observe a heat change with EH3 in the calorimeter. On the other hand, both NPF motifs of Stonin2 are required for a tight binding. Mutations of NPF1 or 2 to triple alanines either significantly reduce the affinity (NPF2-AAA) or complete abrogate the interaction (NPF1-AAA).
Eps15 Figure 2

Figure 2: Only Eps15 EH2 is responsible for high affinity binding to the Stonin2. ITC replots for the interaction of the various EH domains with the NPF region of Stonin2.

Eps15 Figure 3

Figure 3: The presence of both NPF motifs within Stonin2 is required for the high affinity interaction.

We solved the structure of the complex consisting of Eps15-EH2 and the Stonin2 NPF-region in collaboration with Michael Sattler’s group, using NMR spectroscopy (see also (Rumpf et al., 2008a). The structure (Figure 4) shows how both NPF motifs can simultaneously interact with one EH domain. Whereas NPF1 binds into the conserved ligand binding pocket between helices 2 and 3, the second NPF motif inserts into a groove formed by helices 1, 2 and 4. This newly identified pocket is far less conserved and the critical residues that mediate interaction to NPF2 are only present in Eps15 and Eps15R-EH2 domains.

The first binding site resembles previously characterized EH-NPF complexes (de Beer et al., 2000). Only few contacts were observed for residues outside the core NPF motif. Thus, this binding site is more promiscuous when it comes to ligand binding and would interact with a number of ligands with relatively low affinity. In contrast, the second binding site is characterized by a high number of contacts between residues flanking NPF2 and the EH domain. In particular the hydrophobic residues preceding NPF2 (Pro326 and Ile327) as well as two Phe residues (334-335) following the motif are involved in the interaction. These structural observations could be confirmed by a detailed study using binding site mutants, see (Rumpf et al., 2008b). Although this newly identified binding pocket is unique among human EH domains, it is conserved regarding Eps15 proteins throughout evolution.

Figure 4: Structure of the Eps15-EH2-Stonin2 complex. Both NPF motifs of Stonin2 bind into two separate binding pockets on the EH domain. NPF1 inserts into the conserved binding site, NPF2 recognizes a novel, non-conserved hydrophobic groove on the EH domain. Residues that specifically interact with NPF2 and flanking regions are unique to Eps15-EH2 and the related Eps15R-EH2.


Figure 5: Close-up view on the first and second binding site. A) NPF1 binds into the previously reported binding pocket of EH2 and interacts withconserved residues, W169, L165 and L155. The peptide adopts a beta-turn conformation to deeply instert the Pro and Phe residues of NPF1 into the pocket and only few contacts are made by residues outside the NPF core motif. B) NPF2 interacts with residues of helices 2 and 4, a less conserved region of the EH domain. Several residues outside the core NPF motif are involved in binding and increase the specificity among them P327, I328, F334 and F335.