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. Doal functions in the endosomal sorting by its association with Hsel, a component of endosomal sorting complex required for transport (ESCRT) system. The association of Doa1 with Hsel was previously reported to be mediated between PFU domain of Doal and SH3 of Hsel. However, it remains unclear which residues are specifically involved in the interaction. Here we report that Doal/PFU interacts with Hsel/SH3 with a moderate affinity of 5 lLM. Asn-438 of Doal/PFU and Trp-254 of Hsel/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 Doal/PFU:Hsel/SH3 complex. Taken together, our results suggest that hydrogen bonding is a major determinant in the interaction of Doa1/PFU with Hsel/SH3. (C) 2014 Elsevier Inc. All rights reserved.