Dimerisation of the UBA Domain of p62 Inhibits Ubiquitin Binding and Regulates NF-κB Signalling

Jed Long, Thomas P. Garner, Maya J. Pandya, C. Jeremy Craven, Ping Chen, Barry Shaw, Michael P. Williamson, Robert Layfield, Mark S. Searle

Research output: Contribution to journalArticle

67 Scopus citations

Abstract

The ubiquitin (Ub)-binding p62 scaffold protein (encoded by the SQSTM1 gene) regulates a diverse range of signalling pathways leading to activation of the nuclear factor kappa B (NF-κB) family of transcription factors and is an important regulator of macroautophagy. Mutations within the gene encoding p62 are commonly found in patients with Paget's disease of bone and largely cluster within the C-terminal ubiquitin-associated (UBA) domain, impairing its ability to bind Ub, resulting in dysregulated NF-κB signalling. However, precisely how Ub-binding is regulated at the molecular level is unclear. NMR relaxation dispersion experiments, coupled with concentration-dependent NMR, CD, isothermal titration calorimetry and fluorescence kinetic measurements, reveal that the p62 UBA domain forms a highly stable dimer (Kdim ∼ 4-12 μM at 298 K). NMR analysis shows that the dimer interface partially occludes the Ub-binding surface, particularly at the C-terminus of helix 3, making UBA dimerisation and Ub-binding mutually exclusive processes. Somewhat unusually, the monomeric UBA appears to be the biologically active form and the dimer appears to be the inactive one. Engineered point mutations in loop 1 (E409K and G410K) are shown to destabilise the dimer interface, lead to a higher proportion of the bound monomer and, in NF-κB luciferase reporter assays, are associated with reduced NF-κB activity compared with wt-p62.

Original languageEnglish (US)
Pages (from-to)178-194
Number of pages17
JournalJournal of Molecular Biology
Volume396
Issue number1
DOIs
StatePublished - Feb 12 2010

Keywords

  • NMR structural analysis
  • Paget's disease of bone
  • UBA dimerisation
  • ubiquitin
  • ubiquitin-associated domain

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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    Long, J., Garner, T. P., Pandya, M. J., Craven, C. J., Chen, P., Shaw, B., Williamson, M. P., Layfield, R., & Searle, M. S. (2010). Dimerisation of the UBA Domain of p62 Inhibits Ubiquitin Binding and Regulates NF-κB Signalling. Journal of Molecular Biology, 396(1), 178-194. https://doi.org/10.1016/j.jmb.2009.11.032