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Dimerisation of the UBA Domain of P62 Inhibits Ubiquitin Binding and Regulates NF-Κb Signalling

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Dimerisation of the UBA Domain of P62 Inhibits Ubiquitin Binding and Regulates NF-Κb Signalling Author's personal copy doi:10.1016/j.jmb.2009.11.032 J. Mol. Biol. (2010) 396, 178–194 Available online at www.sciencedirect.com Dimerisation of the UBA Domain of p62 Inhibits Ubiquitin Binding and Regulates NF-κB Signalling Jed Long1,2, Thomas P. Garner1,2, Maya J. Pandya3, C. Jeremy Craven3, Ping Chen1,2, Barry Shaw4, Michael P. Williamson3, Robert Layfield4 and Mark S. Searle1,2⁎ 1Centre for Biomolecular The ubiquitin (Ub)-binding p62 scaffold protein (encoded by the SQSTM1 Sciences, University Park, gene) regulates a diverse range of signalling pathways leading to activation Nottingham NG7 2RD, UK of the nuclear factor kappa B (NF-κB) family of transcription factors and is an important regulator of macroautophagy. Mutations within the gene 2School of Chemistry, encoding p62 are commonly found in patients with Paget's disease of bone University Park, and largely cluster within the C-terminal ubiquitin-associated (UBA) Nottingham NG7 2RD, UK domain, impairing its ability to bind Ub, resulting in dysregulated NF-κB 3Department of Molecular signalling. However, precisely how Ub-binding is regulated at the Biology and Biotechnology, molecular level is unclear. NMR relaxation dispersion experiments, coupled Western Bank, with concentration-dependent NMR, CD, isothermal titration calorimetry University of Sheffield, and fluorescence kinetic measurements, reveal that the p62 UBA domain ∼ − μ Sheffield S10 2TN, UK forms a highly stable dimer (Kdim 4 12 M at 298 K). NMR analysis shows 4 that the dimer interface partially occludes the Ub-binding surface, School of Biomedical Sciences, particularly at the C-terminus of helix 3, making UBA dimerisation and University of Nottingham, Ub-binding mutually exclusive processes. Somewhat unusually, the Queen's Medical Centre, monomeric UBA appears to be the biologically active form and the dimer Nottingham NG7 2UH, UK appears to be the inactive one. Engineered point mutations in loop 1 (E409K Received 7 September 2009; and G410K) are shown to destabilise the dimer interface, lead to a higher received in revised form proportion of the bound monomer and, in NF-κB luciferase reporter assays, 11 November 2009; are associated with reduced NF-κB activity compared with wt-p62. accepted 12 November 2009 © 2009 Elsevier Ltd. All rights reserved. Available online 17 November 2009 Keywords: ubiquitin; ubiquitin-associated domain; Paget's disease of bone; Edited by A. G. Palmer III NMR structural analysis; UBA dimerisation Introduction More recently, p62 has been shown to be a critical regulator of the degradation of ubiquitinated pro- 3,4 The p62 protein (encoded by the SQSTM1 gene) teins by macroautophagy. The p62 protein has a domain structure consistent with its participation in functions as a ubiquitin (Ub)-binding scaffold, 5 which regulates a diverse range of signalling path- multiple signalling complexes, including a C- terminal Ub-associated (UBA) domain through ways leading to activation of the nuclear factor 6,7 kappa B (NF-κB) family of transcription factors.1,2 which p62 binds non-covalently to Ub. An important role of p62 is in the control of induced osteoclastogenesis,8 a process dependent on p62's *Corresponding author. E-mail address: ability to regulate RANK-mediated NF-κB signal- [email protected]. ling, probably through the K63-linked ubiquitination Abbreviations used: UBA domain, ubiquitin-associated of TRAF6 and other signalling intermediates. Ac- domain; mUBA, monomeric UBA; Ub, ubiquitin; mUb, cordingly, mutations affecting the SQSTM1 gene are monomeric ubiquitin; NF-κB, nuclear factor kappa B; commonly found in patients with the skeletal PDB, Paget's disease of bone; CPMG, Carr–Purcell– disorder Paget's disease of bone (PDB), a chronic Meiboom–Gill; HSQC, heteronuclear single quantum condition characterised by excessive bone turnover coherence; CSP, chemical shift perturbation; NOE, nuclear leading to bone expansion, structural weakness, Overhauser enhancement; NOESY, nuclear Overhauser deformity and pain.9 The vast majority of the PDB- enhancement spectroscopy; GST, glutathione S-transferase. associated mutations identified to date cluster within 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved. Author's personal copy p62 UBA Domain Dimerisation Inhibits Ub-Binding 179 the UBA domain, impairing p62's ability to bind Ub p62 UBA domain forms a highly stable symmetrical and resulting in dysregulated NF-κB signalling.10,11 dimer. We show that the Ub-binding surface of the The structure–function relationship between the monomeric UBA and the dimer interface partially sites of PDB-associated mutations in the p62-UBA overlap such that dimerisation and Ub-binding are domain and their effects on protein folding, mutually exclusive events, with the former strongly Ub-recognition and dysregulation of NF-κB signal- modulating the latter. Somewhat unusually, the ling are still largely unclear, as are the mechanisms monomeric UBA is the biologically active form, that regulate normal Ub-recognition. Human p62 is whilst the dimer is inactive with a role in regulating a 440-residue protein, and the isolated C-terminal levels of signal activation. We demonstrate this polypeptide sequence (residues 387–436) forms a using a number of point mutations in loop 1, which compact three-helix bundle,7 with close structural destabilise the dimer interface and lead to a higher – homology to other UBA and CUE domains.12 20 All proportion of Ub-bound UBA domain than ob- have been shown to bind non-covalently to mono- served for wt-UBA. The effects of these mutations on meric Ub (mUb) through a common hydrophobic NF-κB signalling, assessed in luciferase reporter patch on the β-sheet surface of Ub.21,22 In contrast, assays, support the role of dimer formation in the UBA domains, which have low sequence modulating the levels of signal activation. homology, show diverse modes of interaction with wide-ranging binding affinities and specificities for both a single Ub motif and for the more compact 23 Results K48-linked Ub2, where the UBA domain is engaged simultaneously with binding surfaces on each of the Ub motifs.17,19 Biophysical analysis supports formation of a Although the majority of UBA domains appear to p62 UBA dimer bind Ub as monomers, the UBA-related CUE domain of Vps9p recognises Ub through an Titrations of p62-UBA with Ub at NMR concen- asymmetric domain-swapped dimer.24 More re- trations of protein (0.2−1 mM) have revealed a cently, dimers have been characterised for the binding mechanism involving a UBA conformer UBA domains of c-Cbl and Cbl-b involving hydro- that is sparsely populated in the absence of Ub, phobic surface interactions at the interface of helices leading to a complex binding process involving both – 2 and 3.25 27 The role of c-Cbl and Cbl-b as scaffold slow- and fast-exchange steps.29 The minor con- proteins, coupled with their capacity to interact former could not be observed directly in hetero- with different downstream signalling proteins, nuclear single quantum coherence (HSQC) spectra; suggests that dimerisation provides a mechanism however, NMR transverse relaxation techniques are for an expanded repertoire of Cbl-dependent suited to detecting the effects of low populations of signals recruited to receptor-tyrosine kinases. Al- different species at equilibrium that are in chemical though, in the case of the c-Cbl homodimer, Ub exchange.31–37 15N relaxation dispersion data col- recognition does not appear to be its primary lected with 0.8 mM p62 UBA at 600 MHz showed function,25 the dimerisation and activation of the strong dispersion effects at 310 K, although these Ub protein ligase Cbl-b are Ub-mediated.26 Within were less evident at lower temperatures where the context of the yeast proteins Rad23 and Ddi1, exchange rates were slower. Well-resolved cross identified in the regulation of the metaphase– peaks were observed for 43 of the 50 residues. Flat anaphase transition of cells through mitosis, the relaxation dispersion profiles were evident for 25 UBA domains were found to be essential for residues; however, the remaining 18 residues homodimerisation of Rad23 and for the heterodi- exhibited clear dispersion effects due to exchange merisation of Rad23 and Ddi1. In both cases, the with a minor conformer. UBA domains bind Ub, with a regulatory role for To identify an appropriate model for data analysis, Rad23 dimerisation in the control of Ddi function including the possibility of a monomer–dimer suggested by the fact that dimerisation appears to equilibrium, we used a number of other biophysical block Ub binding.28 In contrast, the UBA domains techniques sensitive to population shifts at micro- of the human homologue of Rad23 (hHR23a), molar concentrations. The helix-rich p62 UBA which stimulates DNA repair mechanisms, show domain gives a CD spectrum with double minima no evidence of dimerisation.16,29 observed at 208 and 222 nm. The temperature The p62 UBA domain, in line with many other dependence of the signal at 222 nm results in a UBA domains, appears to have a low affinity for Ub reversible sigmoidal unfolding transition (Fig. 1a); when measured by NMR at protein concentrations however, the melting curves show a distinct concen- ∼ − μ 30 in the millimolar range (Kd 500 700 M). The tration dependence of the Tm, as evident from the affinity appears not to be significantly different data collected at concentrations of 3 and 82 μM μ when binding to K48-linked Ub2 and longer chains; shown in Fig. 1a. Above 20 M, the Tm remains neither is there any evidence for apparent chain- invariant with [UBA] at 345 K but decreases by 6 K at linkage specificity. We report NMR studies, includ- the low-concentration end of the range with a ing relaxation dispersion analysis, together with CD, transition midpoint at around 10 μM(Fig. 1b). The isothermal titration calorimetry (ITC) and kinetic data are consistent with self-association modulating fluorescence measurements, which reveal that the the thermal stability of the UBA domain.
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