Structural Basis for Ubiquitin Recognition by a Novel Domain from Human Phospholipase A2-activating Protein

Abstract

Ubiquitin (Ub) is an essential modifier conserved in all eukaryotes from yeast to human. Phospholipase A 2 -activating protein (PLAA), a mammalian homolog of yeast DOA1/UFD3, has been proposed to be able to bind with Ub, which plays important roles in endoplasmic reticulum-associated degradation, vesicle formation, and DNA damage response. We have identified a core domain from the PLAA family ubiquitin-binding region of human PLAA (residues 386 -465, namely PFUC) that can bind Ub and elucidated its solution structure and Ubbinding mode by NMR approaches. The PFUC domain possesses equal population of two conformers in solution by cis/ trans-isomerization, whereas the two isomers exhibit almost equivalent Ub binding abilities. This domain structure takes a novel fold consisting of four ␤-strands and two ␣-helices, and the Ub-binding site on PFUC locates in the surface of ␣2-helix, which is to some extent analogous to those of UBA, CUE, and UIM domains. This study provides structural basis and biochemical information for Ub recognition of the novel PFU domain from a PLAA family protein that may connect ubiquitination and degradation in endoplasmic reticulum-associated degradation.The eukaryotic secreted proteins are translocated into the endoplasmic reticulum after synthesis in cytosol. Misfolded or abnormally assembled proteins should be targeted for degradation through the endoplasmic reticulum-associated degradation (ERAD) 3 pathway (1, 2). This pathway involves many molecular steps: unfolded protein response in the endoplasmic reticulum lumen, retrotranslocation back into the cytosol, ubiquitin (Ub) conjugation, delivery of ubiquitinated proteins to proteasome, and degradation of the substrates by proteases (3, 4).A yeast protein DOA1/UFD3 has been shown to bind to CDC48 by both indirect and direct ways (5-7), suggesting that DOA1 may be involved in ERAD. Evidence indicates that DOA1 directly competes with UFD2 at the same docking site on CDC48, which determines whether a substrate is multiubiquitinated and routed to the proteasome for degradation or deubiquitinated and released for other purposes (8). The direct interaction between DOA1 and Ub was suggested by recent studies (7, 9). Moreover, DOA1 also plays roles in the monoubiquitination of histone H2B and proliferating cell nuclear antigen (10) and in sorting ubiquitinated membrane proteins into multivesicular bodies (11).The mammalian homolog of DOA1 is called phospholipase A 2 -activating protein (PLAA), which can bind to P97/VCP (a CDC48 homolog) with its C-terminal domain PUL (7). Having high sequence similarity (31% identity) with DOA1, PLAA is proposed to possess similar function of DOA1. Like DOA1, PLAA has an N-terminal WD40 domain with yet unknown function. The central region of PLAA contains a putative PLAA family ubiquitin-binding (PFU) domain, which is supposed to bind with Ub as observed in yeast DOA1 (7). Although the mechanism underlying the function of PLAA remains unclear, Ub binding of PLAA might be the central role that connects ubiquitin...

WD40 Repeat Propellers Define a Ubiquitin-Binding Domain that Regulates Turnover of F Box Proteins

“…Ub binding was confirmed using NMR HSQC experiments that showed specific chemical shift perturbations within 15 N-Ub in the presence of unlabelled His-tagged Doa1 β-Prp (Figure 1C and see Figure S1A available online). Quantifying chemical shift perturbations as a function of increasing Doa1 β-Prp concentrations indicated a K d of ~220 μM for mono-Ub (Figure 1D), a moderate affinity within the range of previously described UBDs (Dikic et al, 2009; Harper and Schulman, 2006; Hurley et al, 2006) and which is tighter than the >1 mM K d reported for the mammalian Doa1/Ufd3 PFU domain (Fu et al, 2009). Additional HSQC experiments were performed with 15 N-labeled Doa1 β-Prp, which showed a well-dispersed spectrum consistent with the predicted β-stranded propeller structure.…”

“…The domains of Doa1 (Figure 1A) previously identified by homology and crystallographic studies include a C-terminal armadillo repeat domain (PUL; residues 465–715), a central PFU domain that binds Ub (320–450), and an N-terminal WD40 repeat-containing region predicted to form a β-Prp (residues 1–300) (Fu et al, 2009; Qiu et al, 2010). We performed a series of binding experiments using epitope-tagged fragments of Doa1 containing both the β-Prp and PFU domains or a fragment of the β-Prp alone.…”

“…To better understand Doa1 function, we investigated how Doa1 associates with Ub. Previous mutagenesis and NMR structural studies have shown that the central PFU domain (PLAA family Ub-binding domain) of PLAA binds Ub with near 1 mM K d (Fu et al, 2009; Mullally et al, 2006). However, this interaction is not required for function, indicating other UBDs are present (Ren et al, 2008).…”

Computational modeling of Repeat1 region of INI1/hSNF5: An evolutionary link with ubiquitin

“…The structural core (Rpt1) shows striking similarity to PFU, the PLAA family of Ubiquitin binding domain. PFU domain has been identified as an ubiquitin‐associated domain containing (α + β) topology . Currently, the exact function of either the PFU domain or other proteins with same or similar fold is unknown.…”

Solution structure of the N‐terminal domain of DC‐UbP/UBTD2 and its interaction with ubiquitin

“…On the other hand, when 15 N-labeled Ub is titrated with unlabeled DC-UbP_N, it also causes chemical shift changes of the amide resonances of some residues on Ub [ Fig. 2(E UIM, 13 ZnF-UBP, 15 and PFU, 14 have been identified to bind specifically with Ub and its analogs with variable affinities within micromolar to millimolar scale. 4 Interestingly, SH3, a canonical domain binding with proline-rich motifs found in diverse signal transduction proteins, has also been identified to bind with Ub.…”