title: Structure-based non-canonical amino acid design to covalently crosslink an antibody-antigen complex.
abstract: Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody-antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Jianqing
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: Drew
authors.initials: D
authors.lastname: Tack
authors.affiliation: Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA; Applied Research Laboratories, The University of Texas at Austin, Austin, TX 78758, USA.
authors.forename: Randall A
authors.initials: RA
authors.lastname: Hughes
authors.affiliation: Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA; Applied Research Laboratories, The University of Texas at Austin, Austin, TX 78758, USA. Electronic address: ellingtonlab@gmail.com.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA. Electronic address: jgray@jhu.edu.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Gray
mesh_terms: D000907:Antibodies, Bacterial / Q000737:chemistry*; D000942:Antigens, Bacterial / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry*; D003198:Computer Simulation; D003432:Cross-Linking Reagents / Q000737:chemistry; D004926:Escherichia coli; D007980:Levodopa / Q000737:chemistry; D008958:Models, Molecular; D011485:Protein Binding; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D012984:Software
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D003432:Cross-Linking Reagents; C030325:anthrax toxin; D007980:Levodopa
keywords: Antibody; Binding affinity; Computer-aided design; Crosslinking; Non-canonical amino acid; Rosetta; Structure-based design; l-DOPA