Abstract
The hexameric ATPase p97 has been implicated in diverse cellular processes through interactions with many different adaptor proteins at its N-terminal domain. Among these, the Ufd1-Npl4 heterodimer is a major adaptor, and the p97-Ufd1-Npl4 complex plays an essential role in endoplasmic reticulum-associated degradation (ERAD), acting as a segregase that translocates the ubiquitinated client protein from the ER membrane into the cytosol for proteasomal degradation. We determined the crystal structure of the complex of the N-terminal domain of p97 and the SHP box of Ufd1 at a resolution of 1.55 Å. The 11-residue-long SHP box of Ufd1 binds at the far-most side of the Nc lobe of the p97 N domain primarily through hydrophobic interactions, such that F225, F228, N233 and L235 of the SHP box contact hydrophobic residues on the surface of the p97 Nc lobe. Mutating these key interface residues abolished the interactions in two different binding experiments, isothermal titration calorimetry and co-immunoprecipitation. Furthermore, cycloheximide chase assays showed that these same mutations caused accumulation of tyrosinase-C89R, a well-known ERAD substrate, thus implying decreased rate of protein degradation due to their defects in ERAD function. Together, these results provide structural and biochemical insights into the interaction between p97 N domain and Ufd1 SHP box.
Highlights
The capacity of p97 to engage in diverse cellular processes is mediated by interacting with a variety of adaptor proteins at its N-terminal domain and C-terminal tail
Far more adaptors bind the N-terminal domain, and five different p97N-binding modules included in these adaptors have been identified to date: UBX, UBD, VIM, VBM and SHP box [9,10]
In the present study, the SHP box was observed to bind at the far-most side of the Nc lobe, which is distant from the inter-subdomain cleft where the other four p97N-binding modules bind, and this observation was well consistent with a recent study [33]
Summary
P97, known as VCP (valosin-containing protein), is a hexameric ATPase of type II AAA+ family [1]. P47 has been implicated in cellular processes accompanying membrane fusion events, such as post-mitotic Golgi reassembly [11], whereas the Ufd1-Npl heterodimer was initially characterized in ER-associated degradation [12,13]. The two major adaptors, Ufd1-Npl and p47, share a common mechanism of bipartite binding to p97, in that both adaptors contain a SHP box and that the Npl UBD is similar to p47 UBX in the binding site as well as in the overall fold [23,24]. To gain insights into the assembly principle and the molecular mechanism of p97-Ufd1-Npl segregase complex, we initiated a series of structural and biochemical studies on this complex In this initial report, we present the crystal structure of the complex between the p97 N domain and the Ufd SHP box at a high resolution of 1.55 Å. Based on the complete atomic details of this interaction, we further explored the functional implications of this complex using binding experiments and cell-based ER-associated degradation assays
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