Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 396, Issue 1, Pages 178-194Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2009.11.032
Keywords
ubiquitin; ubiquitin-associated domain; Paget's disease of bone; NMR structural analysis; UBA dimerisation
Categories
Funding
- Biotechnology and Biological Sciences Research Council
- Engineering and Physical Sciences Research Council
- School of Chemistry at Nottingham
- National Association for the Relief of Paget's Disease
- Overseas Research Scholarship
- Biotechnology and Biological Sciences Research Council [BB/D015308/1] Funding Source: researchfish
- BBSRC [BB/D015308/1] Funding Source: UKRI
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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-kappa B) 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-kappa B 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 (K-dim similar to 4-12 mu M 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-kappa B luciferase reporter assays, are associated with reduced NF-kappa B activity compared with wt-p62. (C) 2009 Elsevier Ltd. All rights reserved.
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