Evolution of a carbohydrate binding module into a protein-specific binder.
A carbohydrate binding module, CBM4-2, derived from the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures.
Gunnarsson
Lavinia Cicortas
LC
Department of Immunotechnology, Lund University, P.O. Box 7031, S-220 07 Lund, Sweden.
Dexlin
Linda
L
Karlsson
Eva Nordberg
EN
Holst
Olle
O
Ohlin
Mats
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D045245:Rhodothermus / Q000201:enzymology; D013379:Substrate Specificity; D014990:Xylans / Q000737:chemistry*; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D007074:Immunoglobulin G; D019151:Peptide Library; D014990:Xylans; D014995:Xylosidases