AI-designed prion-capping proteins provide evidence that prion fibril ends are replication-competent surfaces that contribute to prion seeding activity and infectivity.

End-elongation of amyloid fibrils is a prevailing theory to explain prion replication, but direct experimental evidence for this phenomenon is limited by the lack of research tools. To serve as molecular probes for prion fibril termini, here, we designed protein binders against high-resolution structures of infectious prion fibrils using a diffusion-based design model. By generating protein scaffolds around short β-strand segments from terminal prion rungs, we designed β-hairpin-interfacing proteins that cap prion fibrils, which we termed PRICAPs. We validated that PRICAPs exhibit binding to prion fibril termini and confirmed the role of prion fibril ends in replication by demonstrating that PRICAPs inhibit prion seeding activity and attenuate prion replication in organotypic cerebellar slice cultures. Collectively, these findings describe a class of prion-capping protein that can be used to probe prion fibril termini and verify that these surfaces contribute to prion replication and infectivity. IMPORTANCE: Prions replicate by templating the misfolding of native proteins; however, direct evidence identifying the precise sites of replication has remained limited. Here, we address this gap by developing a new class of rationally designed protein tools that specifically bind and cap the ends of infectious prion fibrils. Using these probes, we demonstrate that fibril termini are replication-competent surfaces required for prion seeding and propagation. By inhibiting these sites, our designed proteins markedly reduce prion replication in a disease-relevant