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

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 se...

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Autores principales: Kim, Dae Young, Kandalaft, Hiba, Lowden, Michael J., Yang, Qingling, Rossotti, Martin A., Robotham, Anna, Kelly, John F., Hussack, Greg, Schrag, Joseph D., Henry, Kevin A., Tanha, Jamshid
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10641266/
https://www.ncbi.nlm.nih.gov/pubmed/37742917
http://dx.doi.org/10.1016/j.jbc.2023.105278
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author Kim, Dae Young
Kandalaft, Hiba
Lowden, Michael J.
Yang, Qingling
Rossotti, Martin A.
Robotham, Anna
Kelly, John F.
Hussack, Greg
Schrag, Joseph D.
Henry, Kevin A.
Tanha, Jamshid
author_facet Kim, Dae Young
Kandalaft, Hiba
Lowden, Michael J.
Yang, Qingling
Rossotti, Martin A.
Robotham, Anna
Kelly, John F.
Hussack, Greg
Schrag, Joseph D.
Henry, Kevin A.
Tanha, Jamshid
author_sort Kim, Dae Young
collection PubMed
description 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(H)s, 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(H)H 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.
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spelling pubmed-106412662023-11-14 Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages Kim, Dae Young Kandalaft, Hiba Lowden, Michael J. Yang, Qingling Rossotti, Martin A. Robotham, Anna Kelly, John F. Hussack, Greg Schrag, Joseph D. Henry, Kevin A. Tanha, Jamshid J Biol Chem Research Article 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(H)s, 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(H)H 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. American Society for Biochemistry and Molecular Biology 2023-09-22 /pmc/articles/PMC10641266/ /pubmed/37742917 http://dx.doi.org/10.1016/j.jbc.2023.105278 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Kim, Dae Young
Kandalaft, Hiba
Lowden, Michael J.
Yang, Qingling
Rossotti, Martin A.
Robotham, Anna
Kelly, John F.
Hussack, Greg
Schrag, Joseph D.
Henry, Kevin A.
Tanha, Jamshid
Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages
title Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages
title_full Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages
title_fullStr Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages
title_full_unstemmed Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages
title_short Sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages
title_sort sequence tolerance of immunoglobulin variable domain framework regions to noncanonical intradomain disulfide linkages
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10641266/
https://www.ncbi.nlm.nih.gov/pubmed/37742917
http://dx.doi.org/10.1016/j.jbc.2023.105278
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