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

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Prion diseases stem from misfolding of the cellular prion protein (PrP^C) into a disease-associated form (PrP^Sc).
• Conformation-specific antibodies can differentiate between these PrP forms.

Purpose of the Study:

• To identify specific mutations in PrP that alter its interaction with anti-PrP antibodies.
• To map the binding interfaces of conformation-specific antibodies with PrP at a residue level.

Main Methods:

• Utilized mutational scanning via cell-surface display to screen 1341 PrP single point mutants.
• Employed single-molecule real-time gene sequencing to quantify mutant enrichment and binding affinity changes.
• Analyzed mutant enrichment data to determine the impact of mutations on antibody binding.

Main Results:

• Identified mutations diminishing antibody binding, revealing key contact residues for antibodies like ICSM18 and D18.
• Mapped antibody binding sites, including discontinuous residues in PrP helix 1 for D18.
• Found that antibody 6H4 binding depends on residues involved in structural rearrangements, suggesting its recognition of both PrP forms.

Conclusions:

• Mutational scanning coupled with deep sequencing offers high-resolution mapping of protein-protein interaction interfaces.
• This approach quantitatively measures the impact of mutations on binding affinity, crucial for understanding PrP structure-function relationships.
• The findings provide insights into antibody specificities and the conformational changes underlying prion diseases.