Keywords:

Alanine, Amino Acid Sequence, Amino Acid Substitution, Binding Sites, Catalysis, Cell Line, Conserved Sequence, Crystallography, X-Ray, DNA-Binding Proteins, Escherichia coli, Gene Deletion, Glutathione Transferase, Humans, Hydrogen Bonding, Intracellular Signaling Peptides and Proteins, Kidney, Models, Molecular, Molecular Sequence Data, NF-kappa B, Nuclear Proteins, Polyubiquitin, Precipitin Tests, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Static Electricity, Substrate Specificity, TNF Receptor-Associated Factor 6, Tumor Necrosis Factor alpha-Induced Protein 3, Ubiquitin, Ubiquitin-Protein Ligases, Ubiquitination

Abstract:

<p>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.</p>