Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages

Dae Young Kim; Hiba Kandalaft; Michael J Lowden; Qingling Yang; Martin A Rossotti; Anna Robotham; John F Kelly; Greg Hussack; Joseph D Schrag; Kevin A Henry; Jamshid Tanha

doi:10.1016/j.jbc.2023.105278

Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages

J Biol Chem. 2023 Nov;299(11):105278. doi: 10.1016/j.jbc.2023.105278. Epub 2023 Sep 22.

Authors

Affiliations

¹ Life Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, Ontario, Canada.
² Life Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Montréal, Quebec, Canada.
³ Life Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, Ontario, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
⁴ Life Sciences Division, Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, Ontario, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada. Electronic address: jamshid.tanha@nrc-cnrc.gc.ca.

Abstract

Most immunoglobulin (Ig) domains bear only a single highly conserved canonical intradomain, inter-β-sheet disulfide linkage formed between Cys23-Cys104, and incorporation of rare noncanonical disulfide linkages at other locations can enhance Ig domain stability. Here, we exhaustively surveyed the sequence tolerance of Ig variable (V) domain framework regions (FRs) to noncanonical disulfide linkages. Starting from a destabilized V_H domain lacking a Cys23-Cys104 disulfide linkage, we generated and screened phage-displayed libraries of engineered V_Hs, bearing all possible pairwise combinations of Cys residues in neighboring β-strands of the Ig fold FRs. This approach identified seven novel Cys pairs in V_H FRs (Cys4-Cys25, Cys4-Cys118, Cys5-Cys120, Cys6-Cys119, Cys22-Cys88, Cys24-Cys86, and Cys45-Cys100; the international ImMunoGeneTics information system numbering), whose presence rescued domain folding and stability. Introduction of a subset of these noncanonical disulfide linkages (three intra-β-sheet: Cys4-Cys25, Cys22-Cys88, and Cys24-Cys86, and one inter-β-sheet: Cys6-Cys119) into a diverse panel of V_H, V_L, and V_HH domains enhanced their thermostability and protease resistance without significantly impacting expression, solubility, or binding to cognate antigens. None of the noncanonical disulfide linkages identified were present in the natural human V_H repertoire. These data reveal an unexpected permissiveness of Ig V domains to noncanonical disulfide linkages at diverse locations in FRs, absent in the human repertoire, whose presence is compatible with antigen recognition and improves domain stability. Our work represents the most complete assessment to date of the role of engineered noncanonical disulfide bonding within FRs in Ig V domain structure and function.

Keywords: disulfide; immunoglobulin variable domain; phage display; protein engineering; protein stability; single-domain antibody (sdAb, nanobody).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Cell Surface Display Techniques
Escherichia coli / genetics
Humans
Immunoglobulin Variable Region* / chemistry
Immunoglobulin Variable Region* / genetics
Immunoglobulin Variable Region* / metabolism
Protein Domains / genetics
Protein Folding

Substances

Immunoglobulin Variable Region