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J. Biol. Chem., Vol. 283, Issue 9, 5427-5440, February 29, 2008

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Ubiquitin Recognition by the Ubiquitin-associated Domain of p62 Involves a Novel Conformational Switch^*

Jed Long, Thomas R. A. Gallagher, James R. Cavey, Paul W. Sheppard^¶, Stuart H. Ralston^||, Robert Layfield¹, and Mark S. Searle²

From the School of Chemistry, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, School of Biomedical Sciences, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, ^¶BIOMOL International LP, Palatine House, Matford Court, Exeter EX2 8NL, and ^||Rheumatic Diseases Unit, University of Edinburgh, Western General Hospital, Edinburgh EH2 2XU, United Kingdom

The p62 protein functions as a scaffold in signaling pathways that lead to activation of NF-B and is an important regulator of osteoclastogenesis. Mutations affecting the receptor activator of NF-B signaling axis can result in human skeletal disorders, including those identified in the C-terminal ubiquitin-associated (UBA) domain of p62 in patients with Paget disease of bone. These observations suggest that the disease may involve a common mechanism related to alterations in the ubiquitin-binding properties of p62. The structural basis for ubiquitin recognition by the UBA domain of p62 has been investigated using NMR and reveals a novel binding mechanism involving a slow exchange structural reorganization of the UBA domain to a "bound" non-canonical UBA conformation that is not significantly populated in the absence of ubiquitin. The repacking of the three-helix bundle generates a binding surface localized around the conserved Xaa-Gly-Phe-Xaa loop that appears to optimize both hydrophobic and electrostatic surface complementarity with ubiquitin. NMR titration analysis shows that the p62-UBA binds to Lys⁴⁸-linked di-ubiquitin with 4-fold lower affinity than to mono-ubiquitin, suggesting preferential binding of the p62-UBA to single ubiquitin units, consistent with the apparent in vivo preference of the p62 protein for Lys⁶³-linked polyubiquitin chains (which adopt a more open and extended structure). The conformational switch observed on binding may represent a novel mechanism that underlies specificity in regulating signalinduced protein recognition events.

Received for publication, June 15, 2007 , and in revised form, November 16, 2007.

^* This work was supported by a grant from the Biotechnology and Biological Sciences Research Council of the UK (to M. S. S. and R. L.), in part by grants from the Arthritis Research Campaign (to S. H. R.), the Medical Research Council John Wheale Bequest (to S. H. R. and R. L.), and AstraZeneca (to T. R. A. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 2jy7, 2jy8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence may be addressed: Tel.: 44-115-823-0107; E-mail: robert.layfield{at}nottingham.ac.uk. ² To whom correspondence may be addressed. Tel.: 44-115-951-3567; E-mail: mark.searle{at}nottingham.ac.uk.

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