Detailed Structural Insights into the p97-Npl4-Ufd1 Interface

Abstract

The AAA ATPase, p97, achieves its versatility through binding to a wide range of cofactor proteins that adapt it to different cellular functions. The heterodimer UN (comprising Ufd1 and Npl4) is an adaptor complex that recruits p97 for numerous tasks, many of which involve the ubiquitin pathway. Insights into the structural specificity of p97 for its UN adaptor are currently negligible. Here, we present the solution structure of the Npl4 "ubiquitin-like" domain (UBD), which adopts a ␤-grasp fold with a 3 10 helical insert. Moreover we performed a chemical shift perturbation analysis of its binding surface with the p97 N domain. We assigned the backbone amides of the p97 N domain and probed both its reciprocal binding surface with Npl4 UBD and its interaction with the p97-binding region of Ufd1. NMR data recorded on a 400-kDa full-length UN-hexamer p97 complex reveals an identical mode of interaction. We calculated a structural model for the p97 N-Npl4 UBD complex, and a comparison with the p97-p47 adaptor complex reveals subtle differences in p97 adaptor recognition and specificity.The AAA ATPase p97 represents ϳ1% of total cytosolic protein and is recruited by a myriad of adaptors to provide energy and/or mechanical force for wide-ranging cellular processes (for a recent review, see Ref. 1). The homohexameric structure of p97 has now been solved in assorted permutations of nucleotide-bound states and biophysical methods including x-ray crystallography, electron microscopy, and small-angle X-ray scattering (2-6). Each p97 monomer consists of three domains: N, D1, and D2. Six monomers associate together to form a homohexameric ring with a diameter of ϳ160 Å and a central hole of ϳ20 Å. The two ATPase domains of p97, D1 and D2, and by association the N domain, move relative to each other as a result of ATP binding/hydrolysis events, affording the hexamer important mechanical properties (for review, see Ref. 7). In most cases, the N domains form the sites of interaction between p97 and some 40 binding partners, which lend specificity to the cellular actions of p97 via intricately woven protein signaling networks.The p97 adaptor complex comprising the heterodimer Npl4-Ufd1 (UN) (8) has biologically well characterized roles in endoplasmic reticulum-associated degradation (ERAD) 2 (9) and regulated ubiquitin-dependent processing (10, 11) as well as other emerging functions including mitotic spindle disassembly (12). Most, if not all, of these processes involve ubiquitin or its close relations. ERAD is a quality control process whereby incorrectly folded or obsolete proteins in the ER are retrotranslocated through channels in the ER membrane, polyubiquitinated, and dismantled by the proteasome. Regulated ubiquitindependent processing employs the proteasome in more specific protein cleavage events, recognizing monoubiquitin signals on membrane-bound transcription factors and severing them from the membrane thereby activating transcription.Despite extensive biochemical and genetics characterization, structural informatio...

Control of p97 function by cofactor binding

“…Currently, only limited structural information on the SHP box-p97 interaction is available, and the exact binding site of the SHP box on the p97 N domain remains unclear. While NMR data suggest that the binding region of the SHP box of UFD1 on the N domain partially overlaps with the binding site for the UBXL domain of NPL4 [59], pull-down experiments demonstrated that SHP box binding to p97 does not interfere with the binding of UBX/UBXL domains, suggesting different binding sites [50].…”

“…The structure of the UBXL domain of NPL4 has been solved by NMR, and based on chemical shift perturbation analysis a structural model for the N domainÀUBXL complex has been calculated [59] (Fig. 2B).…”

Distinct conformations of the protein complex p97-Ufd1-Npl4 revealed by electron cryomicroscopy

“…2). One of the best-studied adaptors is the heterodimer Ufd1-Npl4, the only essential cofactor in yeast (3)(4)(5)(6), which directs p97 into cellular processes regulated by ubiquitin-proteasome degradation such as endoplasmic reticulum-associated degradation (ERAD) (7)(8)(9)(10), mitotic progression (11,12), replication (13), and transcription factor activation (14,15). In ERAD (16), mitosis (17), and nucleus reformation (12), the p97-Ufd1-Npl4 complex has been shown to recognize ubiquitylated substrates associated with different cellular structures and, with the energy obtained from the binding and/or hydrolysis of ATP, to extract them into the cytosol to be recycled after deubiquitylation or degraded by the proteasome (10,16,18,19).…”

“…Ufd1 comprises an N-terminal domain, structurally similar to the N domain of p97 and contains binding sites for both poly-and mono-ubiquitin (37), and a flexible C-terminal domain. The structure of Npl4 is less well understood although two domains, the N-terminal ubiquitin-like (UBX-like) domain and the C-terminal NZF (zinc finger), have been solved by NMR (6,25). The interaction between Ufd1-Npl4 and p97 has been described as bipartite with two different p97-interacting sites located at the C-terminal domain of Ufd1 and the UBX-like domain of Npl4 (4), and it has been proposed that these sites interact with two different p97 N domains (6).…”

“…The structure of Npl4 is less well understood although two domains, the N-terminal ubiquitin-like (UBX-like) domain and the C-terminal NZF (zinc finger), have been solved by NMR (6,25). The interaction between Ufd1-Npl4 and p97 has been described as bipartite with two different p97-interacting sites located at the C-terminal domain of Ufd1 and the UBX-like domain of Npl4 (4), and it has been proposed that these sites interact with two different p97 N domains (6). We have previously shown that one Ufd1-Npl4 heterodimer binds at the periphery of a p97 hexameric ring to form a complex of ∼640 kDa (5).…”