Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination

• Published in: Journal of molecular biology Vol. 430; no. 3; pp. 337 - 347
• Main Authors: Bailey, Lucas J., Sheehy, Kimberly M., Dominik, Pawel K., Liang, Wenguang G., Rui, Huan, Clark, Michael, Jaskolowski, Mateusz, Kim, Yejoon, Deneka, Dawid, Tang, Wei-Jen, Kossiakoff, Anthony A.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 02.02.2018; Elsevier
• Subjects: antibody engineering; Antigens - chemistry; Antigens - ultrastructure; Cell Cycle Proteins - chemistry; Cell Cycle Proteins - ultrastructure; cryo-EM fiducial mark; Cryoelectron Microscopy - methods; Crystallization - methods; crystallization chaperone; Fab elbow angle; Fab–protein complex; Humans; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - ultrastructure; Models, Molecular; Molecular Chaperones - chemistry; Molecular Chaperones - ultrastructure; Peptide Library; Protein Conformation; Protein Engineering; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - ultrastructure; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - ultrastructure; antibody engineering; Asf1; Fab–protein complex; Fab elbow angle; cryo-EM fiducial mark; IDE; cryo-EM; NTD; crystallization chaperone
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2017.12.012

Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the “elbow” regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab–antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones. [Display omitted] •Fab fragments have proven utility in structure determination of challenging macromolecular systems. In some instances, however, the flexibility in the “elbow” linker between the Fab variable and constant domains significantly reduces the effectiveness of this approach.•To eliminate this flexibility requires the linker region between the constant and variable domains of the Fab to be modified to freeze in a single conformational state without the Fab losing its binding function or stability.•Phage display was used to “cinch-up” the connector between the two domains by removing one residue in the elbow linker and then repacking the region around the removed residue to restore stability.•Two different elbow modifications that met the criteria were identified. The resulting elbow variants showed significant improvement in crystallizability and utility as fiducial markers for cryo-electron microscopy.•The elbow variants eliminate conformational heterogeneity existing in native Fab fragments, expanding their utility in enabling solving structures of challenging macromolecular systems by crystallography or single-particle cryo-electron microscopy.