Abstract
AAA+ ATPase p97/valosin-containing protein (VCP)/Cdc48 is a key player in various cellular stress responses in which it unfolds ubiquitinated proteins to facilitate their degradation by the proteasome. P97 works in different cellular processes using alternative sets of cofactors and is implicated in multiple degenerative diseases. Ubiquitin regulatory X domain protein 1 (UBXD1) has been linked to pathogenesis and is unique amongst p97 cofactors because it interacts with both termini of p97. Its N-domain binds to the N-domain and N/D1 interface of p97 and regulates its ATPase activity. The PUB (peptide:N-glycanase and UBA or UBX-containing proteins) domain binds the p97 C-terminus, but how it controls p97 function is still unknown. Here we present the NMR structure of UBXD1-PUB together with binding studies, mutational analysis, and a model of UBXD1-PUB in complex with the p97 C-terminus. While the binding pocket is conserved among PUB domains, UBXD1-PUB features a unique loop and turn regions suggesting a role in coordinating interaction with downstream regulators and substrate processing
Highlights
The ubiquitin proteasome system (UPS) critically regulates various cellular stress response pathways including ER-associated degradation (ERAD), ribosomal quality control (RQC), DNA damage repair, and autophagy that ensure cellular and protein homeostasis
The Ubiquitin regulatory X domain protein 1 (UBXD1)-PUB domain was previously identified as functional p97 binding unit [19,29], and the construct ranging from residues 150–264 yielded a well-dispersed 15 N-HSQC spectrum (Figure 1A)
The averaged rotational correlation time of τc = 7.9 ns is obtained from the ratio of the 15 N longitudinal (T1 ) and transverse (T2 ) relaxation times of all amide resonances (Figure 1B). τc can be correlated to the molecular weight (MW) of the protein in solution and reveals whether a protein forms multimeric assemblies [57]
Summary
The ubiquitin proteasome system (UPS) critically regulates various cellular stress response pathways including ER-associated degradation (ERAD), ribosomal quality control (RQC), DNA damage repair, and autophagy that ensure cellular and protein homeostasis. A key element of the UPS is the conserved AAA+ ATPase p97 ( called Cdc or VCP for valosin-containing protein) that targets a subset of ubiquitin-modified substrate proteins, extracts them from cellular structures, and unfolds them to facilitate degradation in the proteasome [1,2,3]. The function and activity of p97 is regulated by a large number of cofactor proteins [10]. They include substrate adapters that bind at the N-domain and assist insertion of the substrates into the D1 pore [9,11].
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