Conformation-Dependent Epitopes Recognized by Prion Protein Antibodies Probed Using Mutational Scanning and Deep Sequencing
Autor: | David W. Colby, Kyle M. Doolan |
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Rok vydání: | 2015 |
Předmět: |
DNA
Bacterial Saccharomyces cerevisiae Proteins Prions Mutant Population Saccharomyces cerevisiae Biology Article Deep sequencing Epitope DNA sequencing Epitopes Mice Structural Biology Escherichia coli Animals Protein Interaction Domains and Motifs Binding site DNA Fungal education Molecular Biology Gene Rearrangement education.field_of_study Binding Sites High-Throughput Nucleotide Sequencing Molecular biology Epitope mapping Mutation Cell Adhesion Molecules Epitope Mapping Alpha helix Protein Binding Single-Chain Antibodies |
Zdroj: | Journal of Molecular Biology. 427:328-340 |
ISSN: | 0022-2836 |
Popis: | Prion diseases are caused by a structural rearrangement of the cellular prion protein, PrP(C), into a disease-associated conformation, PrP(Sc), which may be distinguished from one another using conformation-specific antibodies. We used mutational scanning by cell-surface display to screen 1341 PrP single point mutants for attenuated interaction with four anti-PrP antibodies, including several with conformational specificity. Single-molecule real-time gene sequencing was used to quantify enrichment of mutants, returning 26,000 high-quality full-length reads for each screened population on average. Relative enrichment of mutants correlated to the magnitude of the change in binding affinity. Mutations that diminished binding of the antibody ICSM18 represented the core of contact residues in the published crystal structure of its complex. A similarly located binding site was identified for D18, comprising discontinuous residues in helix 1 of PrP, brought into close proximity to one another only when the alpha helix is intact. The specificity of these antibodies for the normal form of PrP likely arises from loss of this conformational feature after conversion to the disease-associated form. Intriguingly, 6H4 binding was found to depend on interaction with the same residues, among others, suggesting that its ability to recognize both forms of PrP depends on a structural rearrangement of the antigen. The application of mutational scanning and deep sequencing provides residue-level resolution of positions in the protein-protein interaction interface that are critical for binding, as well as a quantitative measure of the impact of mutations on binding affinity. |
Databáze: | OpenAIRE |
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