Engineering and Structural Characterization of a Linear Polyubiquitin-Specific Antibody

• Published in: Journal of molecular biology 2012-05, Vol.418 (3-4), p.134-144
• Main Authors: Matsumoto, Marissa L., Dong, Ken C., Yu, Christine, Phu, Lilian, Gao, Xinxin, Hannoush, Rami N., Hymowitz, Sarah G., Kirkpatrick, Donald S., Dixit, Vishva M., Kelley, Robert F.
• Format: Article
• Language: English
• Subjects: Antibodies - chemistry; Antibodies - metabolism; crystal structure; Fluorescent Antibody Technique; immunoglobulin; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - metabolism; Immunoprecipitation; linkage; lysine; Models, Molecular; phage display; Polyubiquitin - chemistry; Polyubiquitin - metabolism; post-translational modification; precipitin tests; Protein Conformation; Protein Engineering; transcription factor NF-kappa B; tumor necrosis factor-alpha; Tumor Necrosis Factor-alpha - chemistry; Tumor Necrosis Factor-alpha - metabolism; ubiquitin; Ubiquitination; Western blotting; X-ray crystallography
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2011.12.053

Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking. We therefore engineered a linear-linkage-specific antibody that is functional in Western blot, immunoprecipitation, and immunofluorescence applications. A crystal structure of the linear-linkage-specific antibody Fab fragment in complex with linear diubiquitin provides molecular insight into the nature of linear chain specificity. We use the antibody to demonstrate that linear polyUb is up-regulated upon tumor necrosis factor α stimulation of cells, consistent with a critical role in nuclear factor κB signaling. This antibody provides an essential tool for further investigation of the function of linear chains. [Display omitted] ► A linear polyUb antibody with exquisite specificity was engineered. ► The antibody recognizes a conformational epitope unique to linear-linked chains. ► Linear chains are up-regulated upon tumor necrosis factor α stimulation of cells.