Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 427, Issue 2, Pages 328-340Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2014.10.024
Keywords
EP1802Y; yeast surface display; epitope mapping; SMRT; discontinuous epitope
Categories
Funding
- National Institutes of Health (NIH)/National Institute of General Medical Sciences Chemistry Biology Interface Program [5 T32 GM008550-17]
- NIH/National Institute of General Medical Sciences Institutional Development Award Program [8 P20 GM103446-13]
- NIH [NS064173]
- University of Delaware Center for Bioinformatics and Computational Biology Core Facility - NIH [5 P20 RR016472-12]
- National Science Foundation [EPS-081425]
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Prion diseases are caused by a structural rearrangement of the cellular prion protein, PrPc, into a disease-associated conformation, PrPsc, 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. (C) 2014 Elsevier Ltd. All rights reserved.
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