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

Our plain-English summary of Structure and interaction of ubiquitin-associated domain of human Fas-associated factor 1, by J. Song, J.K. Park, J.J. Lee, et al (Protein Science, 2009).

The study

• Authors: J. Song, J.K. Park, J.J. Lee, Y.S. Choi, K.S. Ryu, J.H. Kim, E. Kim, K.J. Lee, Y.H. Jeon, E.E. Kim
• Journal: Protein Science
• Published: 2009;18(11):2265-76
• Cited by: 29 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

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 ubiquitin-associated 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 three-helical 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.

In plain English

This was a laboratory study of a protein, not a study of people or of asbestos exposure. In its background section the article cites other researchers' work linking the protein, human Fas-associated factor 1 (FAF1), to asbestos-induced oncogenesis. This study did not test asbestos. The authors determined the crystal structure of one part of FAF1, its ubiquitin-associated (UBA) domain, at 1.73 angstrom resolution. It was a three-helix bundle similar to other UBA domains. Using nuclear magnetic resonance (NMR), they found that the domain bound single ubiquitin and Lys48-linked di-ubiquitin, but not the related proteins SUMO-1 or NEDD8. Binding to di-ubiquitin was stronger: a dissociation constant of 3.5 micromolar measured by calorimetry, against 1.47 millimolar for single ubiquitin measured by NMR. Deleting one amino acid (Thr18) gave a constant of 55 micromolar, more than a 10-fold reduction in di-ubiquitin binding. In human kidney cells (HEK293T), altered versions of the protein bound less polyubiquitin than the normal version. The authors concluded that the results provide structural insight into how this domain recognizes polyubiquitin chains.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory protein-structure study: X-ray crystallography, NMR spectroscopy, isothermal titration calorimetry (ITC), and experiments in cultured cells with mutant proteins
• Who was studied: No people or animals were studied. The subject was the ubiquitin-associated domain of human FAF1 protein (residues 5–47 for crystallography, residues 1–81 for NMR), made in E. coli bacteria. Some binding experiments used cultured human embryonic kidney cells (HEK293T).
• Exposure: No asbestos exposure was studied. The test materials were ubiquitin, Lys48-linked di-ubiquitin, and the ubiquitin-like proteins SUMO-1 and NEDD8, added to the FAF1 domain in the laboratory.
• Main results:
  • The crystal structure of the FAF1 UBA domain, solved at 1.73 angstrom resolution, was a three-helix bundle similar to other UBA domains.
  • The FAF1 UBA domain bound single ubiquitin and di-ubiquitin, but showed no significant binding to SUMO-1 or NEDD8.
  • Binding strength (dissociation constant) for di-ubiquitin measured by ITC was 3.5 micromolar, with 1:1 binding. Single ubiquitin did not show significant binding on ITC.
  • Binding strength for single ubiquitin measured by NMR was 1.47 (plus or minus 0.10) millimolar, weaker than for di-ubiquitin.
  • A mutant missing the amino acid Thr18 had a dissociation constant for di-ubiquitin of 55 micromolar, more than a 10-fold reduction in binding compared with the normal protein.
  • In HEK293T cells, the mutant FAF1 proteins bound less polyubiquitin than the normal protein. The Ile41Asn mutant showed the least binding, and the Thr18 deletion mutant showed a similar reduction.
  • NMR mapping suggested that di-ubiquitin changes shape on binding, with the UBA domain held between the two ubiquitin units in a sandwich-like complex.
• The authors' conclusion: The authors concluded that the FAF1 UBA domain adopts a canonical three-helical bundle, recognizes single ubiquitin and di-ubiquitin but not SUMO-1 or NEDD8, and that the results provide structural insight into how it recognizes polyubiquitin.
• Limitations the authors state:
  • The authors noted that the NMR-based binding value for di-ubiquitin could be erroneous because of signal broadening and disappearance during titration, so they also measured it by ITC.
  • Two residues near a cysteine were excluded from the cross-saturation mapping because of a possible measurement artifact.
  • The authors stated that the role of residues 48–81 still needs further study.

Topics

Medical terms in this study, explained in our Lexicon: Mesothelioma, Crystal Structure.

Related studies

• Factors affecting the interaction of asbestos fibres with mammalian cells: a study using cells in suspension — R.C. Brown, E.A. Sara, et al. (1991)
• Expression of Gab1 lacking the pleckstrin homology domain is associated with neoplastic progression — H. Kameda, J.I. Risinger, et al. (2001)
• Effect of syndecan-1 overexpression on mesenchymal tumour cell proliferation with focus on different functional domains — F. Zong, E. Fthenou, et al. (2010)
• Intercellular interaction dictates cancer cell ferroptosis via NF2-YAP signalling — J. Wu, A.M. Minikes, et al. (2019)
• A human single-domain antibody elicits potent antitumor activity by targeting an epitope in mesothelin close to the cancer cell surface — Z. Tang, M. Feng, et al. (2013)
• BAP1 forms a trimer with HMGB1 and HDAC1 that modulates gene × environment interaction with asbestos — F. Novelli, A. Bononi, et al. (2021)

Browse return to medical research.