Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20.
Nuclear factor kappaB (NF-kappaB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-kappaB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-kappaB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.
Lin
Su-Chang
SC
Department of Biochemistry, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
Chung
Jee Y
JY
Lamothe
Betty
B
Rajashankar
Kanagalaghatta
K
Lu
Miao
M
Lo
Yu-Chih
YC
Lam
Amy Y
AY
Darnay
Bryant G
BG
Wu
Hao
H
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D025821:Polyubiquitin / Q000737:chemistry / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013379:Substrate Specificity; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry; D054875:Ubiquitination
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D009687:Nuclear Proteins; D048029:TNF Receptor-Associated Factor 6; D025801:Ubiquitin; D025821:Polyubiquitin; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D000409:Alanine