Structural insights into ubiquitin recognition and Ufd1 interaction of Npl4

Yusuke Sato,Atsushi Yamagata,Keiji Tanaka,Hikaru Tsuchiya,Shuya Fukai,Yasushi Saeki,Kei Okatsu

doi:10.1038/s41467-019-13697-y

Abstract

Npl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1, Npl4 forms a heterodimer (UN), and functions as a cofactor for the Cdc48 ATPase. Here, we report the crystal structures of yeast Npl4 in complex with Lys48-linked diubiquitin and with the Npl4-binding motif of Ufd1. The distal and proximal ubiquitin moieties of Lys48-linked diubiquitin primarily interact with the C-terminal helix and N-terminal loop of the Npl4 C-terminal domain (CTD), respectively. Mutational analysis suggests that the CTD contributes to linkage selectivity and initial binding of ubiquitin chains. Ufd1 occupies a hydrophobic groove of the Mpr1/Pad1 N-terminal (MPN) domain of Npl4, which corresponds to the catalytic groove of the MPN domain of JAB1/MPN/Mov34 metalloenzyme (JAMM)-family deubiquitylating enzyme. This study provides important structural insights into the polyubiquitin chain recognition by the Cdc48–UN complex and its assembly.

Highlights

Npl[4] is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast
The yNpl4113–580 (E123A K124A E125A) protein yielded high-quality crystals, and its structure was determined at 1.72 Å resolution by the single-wavelength anomalous diffraction (SAD) method using the zinc edge (Table 1)
Some signals derived from SeMet were indistinguishable or not detected, we detected the signals derived from SeMet[1], SeMet[19], and SeMet[26] of the Ubdist and SeMet[1], SeMet[26], and SeMet[30] of the Ubprox

Summary

Introduction

Npl[4] is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Understanding the mechanism of the substrate translocation accompanied with the Ub chain binding and ATP hydrolysis has been a long-standing problem, recent structural and functional studies provided important mechanistic insights into reacting steps of the Cdc48/p97–UN complex[22]. The MPN domain of Npl[4] topologically resembles the catalytic domain of the JAB1/MPN/Mov[34] metalloenzyme (JAMM)-family deubiquitylating enzyme (DUB)[19], which accommodates the C-terminal tail of Ub. the groove corresponding to the catalytic site of JAMM DUBs25,26 has been proposed as a potential Ub-binding site of Npl[4]. Due to the low local resolution, the density for Ufd[1] was not interpreted with the atomic model and the interaction between Npl[4] and the two folded Ub moieties has not been characterized in detail[22]

Methods

Results

Conclusion