Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, South Korea.
Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, South Korea.
Biochem Biophys Res Commun. 2014 Mar 28;446(1):352-7. doi: 10.1016/j.bbrc.2014.02.118. Epub 2014 Mar 4.
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.
酵母 Doa1/Ufd3 是 Cdc48（哺乳动物中的 p97）的衔接蛋白，Cdc48 是一种与内质网相关蛋白降解途径和内体分选到多泡体有关的 AAA 型 ATP 酶。Doa1 通过与 Hse1 的关联在内涵体分选过程中发挥作用，Hse1 是参与运输的内体分选复合物（ESCRT）系统的一个组成部分。先前报道 Doa1 与 Hse1 的关联是由 Doa1 的 PFU 结构域和 Hse1 的 SH3 结构域之间介导的。然而，目前尚不清楚哪些残基参与了相互作用。在这里，我们报告说 Doa1/PFU 与 Hse1/SH3 以 5μM 的中等亲和力相互作用。Doa1/PFU 的 Asn-438 和 Hse1/SH3 的 Trp-254 被发现是相互作用的关键，而通过疏水相互作用参与泛素结合的 Phe-434 则不是。小角度 X 射线散射测量结合分子对接和生化分析得出了 Doa1/PFU:Hse1/SH3 复合物的溶液结构。总之，我们的结果表明氢键是 Doa1/PFU 与 Hse1/SH3 相互作用的主要决定因素。
