title: Molecular determinants of the interaction between Doa1 and Hse1 involved in endosomal sorting.
abstract: Yeast Doa1/Ufd3 is an adaptor protein for Cdc48 (p97 in mammal), an AAA type ATPase associated with endoplasmic reticulum-associated protein degradation pathway and endosomal sorting into multivesicular bodies. Doa1 functions in the endosomal sorting by its association with Hse1, a component of endosomal sorting complex required for transport (ESCRT) system. The association of Doa1 with Hse1 was previously reported to be mediated between PFU domain of Doa1 and SH3 of Hse1. However, it remains unclear which residues are specifically involved in the interaction. Here we report that Doa1/PFU interacts with Hse1/SH3 with a moderate affinity of 5 μM. Asn-438 of Doa1/PFU and Trp-254 of Hse1/SH3 are found to be critical in the interaction while Phe-434, implicated in ubiquitin binding via a hydrophobic interaction, is not. Small-angle X-ray scattering measurements combined with molecular docking and biochemical analysis yield the solution structure of the Doa1/PFU:Hse1/SH3 complex. Taken together, our results suggest that hydrogen bonding is a major determinant in the interaction of Doa1/PFU with Hse1/SH3.
authors.affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, South Korea.
authors.forename: Seungsu
authors.initials: S
authors.lastname: Han
authors.affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, South Korea.
authors.forename: Donghyuk
authors.initials: D
authors.lastname: Shin
authors.affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, South Korea.
authors.forename: Hoon
authors.initials: H
authors.lastname: Choi
authors.affiliation: Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, South Korea. Electronic address: sangholee@skku.edu.
authors.forename: Sangho
authors.initials: S
authors.lastname: Lee
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D054730:Protein Interaction Domains and Motifs; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C099944:DOA1 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; C464563:Hse1 protein, S cerevisiae; D018160:Receptors, Cytoplasmic and Nuclear; D029701:Saccharomyces cerevisiae Proteins
keywords: Doa1; Endosomal sorting; Hse1; Interaction; PFU; SH3