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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...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2023
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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. |
format | Online Article Text |
id | pubmed-10641266 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
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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