Molecular mechanism of thioflavin-T binding to the surface of beta-rich peptide self-assemblies.
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.
Biancalana
Matthew
M
Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.
Makabe
Koki
K
Koide
Akiko
A
Koide
Shohei
S
D052160:Benzothiazoles; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008954:Models, Biological; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013499:Surface Properties; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D052160:Benzothiazoles; D010455:Peptides; D013844:Thiazoles; C009462:thioflavin T; D014443:Tyrosine