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Abstract
A number of small organic molecules have been developed that bind to amyloid fibrils,
a subset of which also inhibit fibrillization. Among these, the benzothiol dye Thioflavin-T
(ThT) has been used for decades in the diagnosis of protein-misfolding diseases and
in kinetic studies of self-assembly (fibrillization). Despite its importance, efforts
to characterize the ThT-binding mechanism at the atomic level have been hampered by
the inherent insolubility and heterogeneity of peptide self-assemblies. To overcome
these challenges, we have developed a minimalist approach to designing a ThT-binding
site in a "peptide self-assembly mimic" (PSAM) scaffold. PSAMs are engineered water-soluble
proteins that mimic a segment of beta-rich peptide self-assembly, and they are amenable
to standard biophysical techniques and systematic mutagenesis. The PSAM beta-sheet
contains rows of repetitive amino acid patterns running perpendicular to the strands
(cross-strand ladders) that represent a ubiquitous structural feature of fibril-like
surfaces. We successfully designed a ThT-binding site that recapitulates the hallmarks
of ThT-fibril interactions by constructing a cross-strand ladder consisting of contiguous
tyrosines. The X-ray crystal structures suggest that ThT interacts with the beta-sheet
by docking onto surfaces formed by a single tyrosine ladder, rather than in the space
between adjacent ladders. Systematic mutagenesis further demonstrated that tyrosine
surfaces across four or more beta-strands formed the minimal binding site for ThT.
Our work thus provides structural insights into how this widely used dye recognizes
a prominent subset of peptide self-assemblies, and proposes a strategy to elucidate
the mechanisms of fibril-ligand interactions.