RT Journal Article SR Electronic T1 Competitive interactions between Eps15 EH domains and intrinsically disordered proteins generate a complex interaction network JF bioRxiv FD Cold Spring Harbor Laboratory SP 2024.09.30.615448 DO 10.1101/2024.09.30.615448 A1 Papagiannoula, Andromachi A1 Vedel, Ida Marie A1 Motzny, Kathrin A1 Tengo, Maud A1 Milles, Sigrid YR 2024 UL http://biorxiv.org/content/early/2024/10/01/2024.09.30.615448.abstract AB Eps15 is one of the earliest arriving proteins at the endocytic site and is of complex architecture: Its N-terminal domain comprises three small Eps15 homology (EH) domains of less than 10 kDa in size, followed by a coiled coil domain and a C-terminal intrinsically disordered region (IDR) of more than 400 residues in length. The EH domains contribute towards the establishment of a complex interaction network within clathrin mediated endocytosis by interacting with Asp-Pro-Phe (NPF) motifs within intrinsically disordered regions of other endocytic proteins. Using NMR spectroscopy, we investigate the molecular origins of binding from the side of the EH domains as well as from the side of the most intrinsically disordered region of the partner Dab2. In addition to NPF binding, we detect a high level of binding promiscuity leading to significant interaction with non-NPF binding sites. This behavior also leads to interactions between Eps15’s EH domains and its own IDR. When all EH domains are expressed in row, as they occur in the wild type full length protein (EH123), EH2 and EH3 tumble together as one entity, while EH1 moves independently. The full EH-domain, comprising EH1, EH2 and EH3, interacts with both Dab2 and Eps15’s IDR at the same time with partially competitive binding sites. These data allow insights into competitive network-like interactions determining the early phases of clathrin mediated endocytosis.Significance Statement Multi-domain proteins are frequent in many biological processes. Rather than just acting like beads on a string, the EH domains of Eps15 engage in interactions, leading to complex and competitive binding with intrinsically disordered regions (IDRs) involved in Eps15 itself as well as of other endocytic proteins. Such interactions are important to understand how the early phases of clathrin mediated endocytosis are regulated and offer ideas for how interactions may take place in other multi-domain/IDR protein systems. The kind of interactions observed are likely to have implications for liquid-liquid phase separation in endocytosis.Competing Interest StatementThe authors have declared no competing interest.