Ubiquitin recognition by the ubiquitin-associated domain of p62 involves a novel conformational switch.
The p62 protein functions as a scaffold in signaling pathways that lead to activation of NF-kappaB and is an important regulator of osteoclastogenesis. Mutations affecting the receptor activator of NF-kappaB 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 Lys 48-linked di-ubiquitin with approximately 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 Lys 63-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.
Long
Jed
J
School of Chemistry, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom.
Gallagher
Thomas R A
TR
Cavey
James R
JR
Sheppard
Paul W
PW
Ralston
Stuart H
SH
Layfield
Robert
R
Searle
Mark S
MS
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010001:Osteitis Deformans / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012441:Saccharomyces cerevisiae; D000071456:Sequestosome-1 Protein; D015398:Signal Transduction / Q000235:genetics; D013329:Structure-Activity Relationship; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D016328:NF-kappa B; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; D025801:Ubiquitin