Molecular mechanism of Thioflavin-T binding to amyloid fibrils

Abstract

Intense efforts to detect, diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of amyloid-specific molecular probes. Since its first description in 1959, the fluorescent dye Thioflavin-T (ThT) has become among the most widely used "gold standards" for selectively staining and identifying amyloid fibrils both in vivo and in vitro. The large enhancement of its fluorescence emission upon binding to fibrils makes ThT a particularly powerful and convenient tool. Despite its widespread use in clinical and basic science applications, the molecular mechanism for the ability of ThT to recognize diverse types of amyloid fibrils and for the dye's characteristic fluorescence has only begun to be elucidated. Here, we review recent progress in the understanding of ThT-fibril interactions at an atomic resolution. These studies have yielded important insights into amyloid structures and the processes of fibril formation, and they also offer guidance for designing the next generation of amyloid assembly diagnostics, inhibitors, and therapeutics.

Figure 1

Common experimental techniques employing ThT. (a) Structure of ThT (top). The two planer segments of ThT whose mutual rotation defines chirality are also shown (bottom). (b) Early histology using Thioflavin-T to stain primary kidney amyloid [7]. (c) TIRF microscopy image of branched glucagon fibrils stained with ThT [21]. (d) Characteristic increase in ThT upon binding to amyloid fibrils. (e) Fibrillization kinetics of increased concentrations of a fibril-forming peptide. The rapid onset of fibrillization induced through seeding is also shown. Images in b and c have been reproduced with permission.

Figure 2

The common structure of fibrils and a structural rationale for fibril-ThT interactions. (a) Cross-β structure of amyloid fibrils, formed from layers of laminated β-sheets. (b) “Channel” model of ThT binding to fibril-like β-sheets. ThT is proposed to bind along surface side-chain grooves running parallel to the long axis of the β-sheet.

Figure 3

Crystal structure of ThT bound to acetylcholinesterase (PDB 2J3Q) [32]. (a) Acetylcholinesterase is shown in cartoon representation, colored gray. All residues within 4 Å of ThT are shown as red sticks, except for Tyr121, which is omitted for clarity. The aromatic residues (Tyr and Trp) forming ThT-binding surfaces similar to that observed in the PSAM are labeled. The carbon, nitrogen and sulfur elements of ThT are in lavendar, blue, and yellow, respectively. (b) The ThT-binding pocket of acetylcholinesterase shown in orthogonal views, with the protein main-chain atoms omitted.

Figure 4

Atomic resolution investigations of ThT binding sites and ThT-β-sheet interactions. (a) and (b) Binding of ThT to the KLVFFAE protofibril probed using molecular dynamics simulations [45]. Only the β-sheet faces of the protofibril are shown, since ThT did not appreciably bind between the laminated β-sheets. (a) Binding of ThT to the “LFA” face of the protofibril β-sheet. The structure of the starting structure is shown at left, with the bottom β-strand labeled to distinguish side chains in the front (large letters) and back (small letters) of the β-sheet. The clustered populations of bound ThT molecules are shown at center. The amino acid identifies of the “LFA” side-chain ladders on this face are shown at right. Side chains are colored blue for positive, red for negative, and black for hydrophobic. The backbone is shown in cartoon representation [45]. (b) Binding of ThT to the “EFVK” β-sheet face of the protofibril, shown as in (a). (c) Crystal structure of the peptide self-assembly mimic (PSAM) scaffold showing the central β-sheet capped by N- and C-terminal domains (PDB 3EC5) [46]. The amino acid identities of the wild-type cross-strand ladders are shown, as well as the designed ladders in the 3-YY/LL, 4-YY/LL, and 5-YY/LL mutants. The relative strength of the interactions of these PSAMs with ThT (as determined by fluorescence intensity and K_d) are indicated below. (d) Magnified view of the ThT-binding β-sheet of the 5-YY/LL PSAM [46]. The amino acid identities of the designed ladders are given in (c). The side chains of the designed residues of the ThT binding site as shown as sticks. These residues and their solvent-exposed surfaces are colored blue for Tyr and green for Leu, while wild-type side chains are in dark gray. The two PEG-400 molecules found near the ThT-binding site are shown as red sticks.. (e) The side-chain conformations of the PSAM ladders implicated in ThT-binding [46]. The ladders are shown looking down the long axis of the PSAM from C- to N-terminus (top) and looking straight down on the β-sheet (bottom). The backbone is depicted in cartoon form, with turns omitted for clarity. At the right is shown a binding orientation for ThT determined by molecular dynamics simulation [50]. The carbon, nitrogen and sulfur elements of ThT are in white, blue, and yellow, respectively. (f) The β-sheet segment of the PSAM used for molecular dynamics simulations of ThT binding [50]. The N- and C-terminal domains were omitted to reduce the computational cost, as these domains have been experimentally demonstrated not to bind ThT. Aromatic residues are shown in green, hydrophobic residues are grey, positively charged residues are blue, and negatively charged residues are red. (g) Binding modes of ThT within an engineered channel [50]. Four clusters are located at: the shadow groove formed by continuous Tyr side chains (Site a); on top of β-strand 3-5 (Site b) and at the two edges of the β-sheet (Sites c & d). ThT molecules are represented by lines. Site a is the most populated and energetically favored of these sites, and precisely corresponds to the engineered ThT-binding mutations. (h) Lateral view of ThT-binding simulation to the PSAM, showing that ThT binds primarily to the designed aromatic-hydrophobic face of the β-sheet [50]. Almost no binding is observed on the opposite charged surface of the β-sheet (each black dot represents a ligand). The N-terminus of the β-sheet is shown as a red ball. Images in all panels have been reproduced with permission.