Conformation-dependent epitopes recognized by prion protein antibodies probed using mutational scanning and deep sequencing

Abstract

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.

Keywords: EP1802Y; SMRT; discontinuous epitope; epitope mapping; yeast surface display.

Figure 1. Deep sequencing subpopulations of PrP mutants sorted for loss of antibody binding affinity identifies critical epitope regions

(A) The initial mutant PrP library displayed on the surface of yeast was labeled with antibodies indicated on left as well as an antibody recognizing a c-myc tag genetically fused to PrP to monitor full-length expression level. The c-myc tag is C-terminal to PrP. The majority of PrP mutants in the library are recognizable by the anti-PrP antibodies, while a small number of mutants have diminished binding to the antibodies, indicated in the polygon labeled “Sort.” (B) Following several rounds of sorting by FACS the resulting sub-populations are primarily composed of mutant PrP clones with diminished binding to the antibodies. (C) Mutations observed by SMRT sequencing of PrP genes contained within populations with diminished antibody binding were mapped by position. (D) Regions of interest where residues containing mutations were enriched in the sorted population relative to wildtype PrP. Calculating the enrichment value accounts for unequal representation of mutations in the initial library and the stringency of sorting.

Figure 2. Substitution distributions of selected residues characterize residue interactions with antibody

The enrichment for each particular substitution is reported for all substitutions arising from a single nucleotide substitution. (A) Epitope residues show enrichment of all or near all substitutions arising from a single nucleotide substitution consistent with the sidechain of these residues directly contacting paratope residues in the antibody (B) Some epitope residues show tolerance of particular substitution, demonstrated by the absence of these substitutions in the sequences obtained from the sorted populations, as a result of physicochemical similarity of the sidechains (C) Positions were only one or a few substitutions show statistically significant enrichment can be the result of particular substitutions that introduce negative interactions (D) Mutations to proline or glycine at many residues were ablative likely by preventing alpha helical secondary structure to develop. Mutations to residues critical to protein folding also led to reduction in affinity.

Figure 3. Experimental validation of critical epitope residues indicates correlation between mutation enrichment and contribution to binding

wild-type (●),N152D (△), W144S (□), K203E (◇), F140S (◆), M212V (▲), and D201G ( ) PrP expressed on the yeast surface were incubated with (A) ICSM18-scFv, (B) D18-scFv, and (C) 6H4-scFv at indicated concentrations. (D) The affinities of each PrP mutant, determined from the 6H4-scFv titration, were plotted as a function of the enrichment observed in the 6H4 sorted sub-library, demonstrating a correlation (Spearman rank correlation coefficient = 0.94, p = 8.3 × 10⁻³) between enrichment and the change in binding affinity. Titration of yeast expressing N152D-PrP did not result in the observance of an increase in fluorescence associated with 6H4-scFv binding preventing calculation of a dissociation constant; N152D as shown represents that the reduction in affinity was greatest among substitutions tested.

Figure 4. Mutational landscape mapped onto native PrP structure reveal structural basis for discontinuous contact residues

(A) The entire mutational landscape arising from single mutant sequences in the sorted sub-libraries were mapped onto the ribbon structure of mouse PrP (PDB ID: 2L39) (B) Magnified view of ribbon structure for epitope region. An arrow is drawn to D201 in the 6H4 condition to highlight its position relative to residues in helix 1. (C) Electron density map for epitope region identify the binding surfaces for each antibody. Despite not contacting adjacent residues, the ICSM18/D18 contact residues form a continuous binding patch along the outside/top of helix 1. The contact residues for 6H4 are composed of residues at the helix 1/ helix 3 interface; arrow is drawn to D201. (D) Electron density map for epitope region where critical contact residues (characterized by statistically significant enrichment of more than 50% of substitutions arising from single nucleotide changes) are indicated in green while all non-epitope residues are indicated in white.