Structure and interaction of ubiquitin‐associated domain of human Fas‐associated factor 1

Abstract

Fas-associated factor (FAF)-1 is a multidomain protein that was first identified as a member of the Fas death-inducing signaling complex, but later found to be involved in various biological processes. Although the exact mechanisms are not clear, FAF1 seems to play an important role in cancer, asbestos-induced mesotheliomas, and Parkinson's disease. It interacts with polyubiquitinated proteins, Hsp70, and p97/VCP (valosin-containing protein), in addition to the proteins of the Fas-signaling pathway. We have determined the crystal structure of the ubiquitinassociated domain of human FAF1 (hFAF1-UBA) and examined its interaction with ubiquitin and ubiquitin-like proteins using nuclear magnetic resonance. hFAF1-UBA revealed a canonical threehelical bundle that selectively binds to mono-and di-ubiquitin (Lys48-linked), but not to SUMO-1 (small ubiquitin-related modifier 1) or NEDD8 (neural precursor cell expressed, developmentally down-regulated 8). The interaction between hFAF1-UBA and di-ubiquitin involves hydrophobic interaction accompanied by a transition in the di-ubiquitin conformation. These results provide structural insight into the mechanism of polyubiquitin recognition by hFAF1-UBA.Additional Supporting Information may be found in the online version of this article The first two authors contributed equally to this work.Abbreviations: hFAF1, human Fas-associated factor 1; Ub, Ubiquitin; di-Ub, Lys48-linked di-ubiquitin; RMSD, root mean square deviation; CSP, chemical shift perturbation.

Complex of Fas-associated Factor 1 (FAF1) with Valosin-containing Protein (VCP)-Npl4-Ufd1 and Polyubiquitinated Proteins Promotes Endoplasmic Reticulum-associated Degradation (ERAD)

“…Human FAF1 was cloned into pFLAG-CMV2 vector to generate pFLAG-CMV2-FAF1. pFLAG-CMV2-FAF1(82-650) and pFLAG-CMV2-FAF1 I41N were prepared as described previously (1,2). FLAG-CMV2-FAF1 constructs used in this study are listed in Fig.…”

“…Cell Extracts and Immunoprecipitation-HEK293T and HeLa cells were grown and maintained in DMEM (Eagle's minimal essential medium) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO 2 . Transfections were carried out with Effectene (Qiagen) transfection reagent in a 1:10 ratio as instructed by the manufacturer.…”

“…Full-length Ufd1 (aa 1-307) and Npl4 (aa 1-608) were cloned into pET-28a and pET-22b (Novagen). GST-fused FAF1 UBA domain (aa 1-81), full-length VCP, and Ufd1-Npl4 complex were purified as described previously (2,33). The full-length FAF1 was overexpressed in Escherichia coli Rosetta-gami (DE3) cells (Novagen), whereas the others were overexpressed in E. coli Rosetta (DE3) cells.…”

“…For imaging Nanogold (ϳ1.5 nm in diameter) on the complex, a Tecnai Titan microscope was used at 300 kV without staining the sample (FEI Co.). To observe both gold particles and protein complex, we used a 618 TFPR 621 ) of FAF1 UBX domain was shortened to AG; FAF1(82-650), FAF1 UBA domain (1-47) was deleted; FAF1 ⌬UBX, FAF1 UBX domain (569 -650) was deleted; FAF1 ⌬UBL1, FAF1 UBL1 domain (100 -174) was deleted; FAF1 ⌬UBL1-2, FAF1 UBL1 domain (100 -174) and UBL2 domain (195-270) were deleted (⌬100 -270); FAF1(TFPR 3 AG) ⌬UBL1, UBL1 domain was deleted in FAF1(TFPR 3 AG) construct; FAF1(TFPR 3 AG) ⌬UBL1-2, UBL1,2 domains were deleted in FAF1(TFPR 3 AG) construct; FAF1 I41N, point mutation of UBA domain at Ile 41 , which is crucial for ubiquitin interaction with N-terminal UBA domain (2). B, constructs of FAF1, VCP, and Npl4-Ufd1 complex used for structural and biochemical studies.…”

Insights into High Affinity Small Ubiquitin-like Modifier (SUMO) Recognition by SUMO-interacting Motifs (SIMs) Revealed by a Combination of NMR and Peptide Array Analysis

“…All SUMO proteins form a ubiquitin-like fold, which contains an ␣-helix and a ␤-sheet. The SIM binds to a surface between the ␣-helix and ␤-sheet and extends the ␤-sheet as either a parallel or antiparallel ␤-strand (17). The known characteristics of SIMs are that they are short (less than 10 amino acid residues) and rich in hydrophobic residues.…”