Structural Basis of Enhanced Crystallizability Induced by a Molecular Chaperone for Antibody Antigen-Binding Fragments. Issue 3 (2nd February 2018)

• Record Type: Journal Article
• Authors: Ereño-Orbea, June; Sicard, Taylor; Cui, Hong; Carson, Jacob; Hermans, Pim; Julien, Jean-Philippe
• Is Part Of: Journal of molecular biology. Volume 430:Issue 3(2018)
• Page Start: 322
• Page End: 336
• Publication Date: 2018-02-02
• Subjects: Abs antibodies -- Ig immunoglobulin -- HC heavy chain -- LC light chain -- Fab antigen-binding fragment -- V variable domain -- C constant domain -- CDR complementarity determining region -- VHH variable heavy-chain Ig domain -- HEK human embryonic kidney cells -- SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis -- BLI biolayer interferometry -- KD equilibrium constant -- kon association constant -- koff dissociation constant -- Tagg aggregation temperature; antibody -- fragment antigen-binding -- X-ray crystallography -- crystallization chaperone; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2017.12.010
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: Monoclonal antibodies constitute one of the largest groups of drugs to treat cancers and immune disorders, and are guiding the design of vaccines against infectious diseases. Fragments antigen-binding (Fabs) have been preferred over monoclonal antibodies for the structural characterization of antibody–antigen complexes due to their relatively low flexibility. Nonetheless, Fabs often remain challenging to crystallize because of the surface characteristics of complementary determining regions and the residual flexibility in the hinge region between the variable and constant domains. Here, we used a variable heavy-chain (VH H) domain specific for the human kappa light chain to assist in the structure determination of three therapeutic Fabs that were recalcitrant to crystallization on their own. We show that this ligand alters the surface properties of the antibody–ligand complex and lowers its aggregation temperature to favor crystallization. The VH H crystallization chaperone also restricts the flexible hinge of Fabs to a narrow range of angles, and so independently of the variable region. Our findings contribute a valuable approach to antibody structure determination and provide biophysical insight into the principles that govern the crystallization of macromolecules. Graphical abstract: Image 1 Highlights: A variable heavy-chain domain (VH H) specifically binds the human kappa light chain. VH H binding decreases the aggregation temperature of Fabs. Fab crystallizability is enhanced by crystal-packing contacts mediated by the VH H. A restricted range of elbow angles is observed for Fabs in complex with VH H. Three therapeutic antibody structures are revealed from Fab-VH H complexes.