The conformational landscape of human transthyretin revealed by cryo-EM

Abstract

Transthyretin (TTR) is a natively tetrameric thyroxine transporter found in blood and cerebrospinal fluid whose misfolding and aggregation causes transthyretin amyloidosis. A rational drug design campaign identified the small molecule tafamidis (Vyndaqel/Vyndamax) as an effective stabilizer of the native TTR fold, and this aggregation inhibitor is regulatory agency-approved for the treatment of TTR amyloidosis. Despite 50 years of structural studies on TTR and this triumph of structure-based drug design, there remains a notable dearth of structural information available to understand ligand binding allostery and amyloidogenic TTR unfolding intermediates. We used single-particle cryo-electron microscopy (cryo-EM) to investigate the conformational landscape of this 55 kiloDalton tetramer in the absence and presence of one or two ligands, revealing inherent asymmetries in the tetrameric architecture and previously unobserved conformational states. These findings provide critical mechanistic insights into negatively cooperative ligand binding and the structural pathways responsible for TTR amyloidogenesis. This study underscores the capacity of cryo-EM to provide new insights into protein structures that have been historically considered too small to visualize and to identify pharmacological targets suppressed by the confines of the crystal lattice, opening uncharted territory in structure-based drug design.

Figure 1.. Unliganded tetrameric TTR adopts multiple asymmetric conformations, as determined by cryo-EM.

(A) A cartoon ribbon representation of TTR (PDB ID: 1ICT) is colored and labeled by protomer. Regions of the β-sheets that form the ligand binding sites (green ovals) are denoted with more saturated coloring. On the right is an orthogonal view where the individual strands of the A-B dimer are labeled. (B) Cryo-EM reconstructions of the canonical, compressed, and frayed states of tetrameric TTR (all resolved to ~3.3 Å resolution), colored by subunit. To emphasize the disordering of the B and D subunits in the frayed state, a two-fold symmetrized semi-transparent TTR density is underlaid beneath the reconstruction. (C) Ribbon representations of β-strands A, D, G, and H, which comprise the binding pocket, are shown with transparent cryo-EM density to highlight the quality of the reconstruction in this region. The different dihedral angles between opposing H β-strand angles in the canonical and compressed states are denoted in black text, and the different pocket volumes corresponding to each ligand binding site are labeled in green text. (D) Licorice representations of the peptide backbones of the canonical TTR conformation determined by cryo-EM (blue) fit to the closest matching crystallography-based atomic model identified in the PDB (ID: 4PVN). (E) Licorice representation of the peptide backbones of the canonical (blue) and compressed (gold) TTR conformations determined by cryo-EM are superimposed to emphasize the compaction of the structure in the compressed state. (F) Ribbon representation of the β-strands shown in (C) are superimposed to emphasize the differences in β-sheet architecture at the dimer-dimer interface.

Figure 2.. Asymmetric binding pose of stilbene tethered to both TTR binding pockets.

(A) Orthogonal views of the cryo-EM reconstructions of the canonical (above) and compressed (below) conformations of TTR, resolved to ~2.7 and ~3.0 Å resolution, respectively, colored by subunit as in Fig. 1. The center image is a cross-sectional view of the cryo-EM density showing the density corresponding to the covalently bound ligand (green). (B) The cryo-EM density of each stilbene moiety in the binding pockets of the canonical state reconstruction is shown as a mesh with the atomic model shown as a stick model. The stilbene covalently attached to Lys15 of chain C in the A-C binding pocket (top panel) is better resolved as a single conformation than the stilbene in the B-D binding pocket (bottom panel). (C) Ribbon representations of β-strands A, D, G, and H along with the stick representation of the bound stilbene ligand as a green stick representation are shown with transparent cryo-EM density to highlight the quality of the reconstruction in this region. The different H β-strand dihedral angles in the canonical and compressed states are denoted. To the right, a cutaway view of the surface representation of the ligand binding pockets with the stilbene ligand rendered a green stick representation is shown, with the different pocket volumes denoted in green text. (D) Ribbon representation of the β-strands shown in (C) are superimposed to emphasize the differences in β-sheet architecture at the dimer-dimer interface.

Figure 3.. Asymmetric single-liganded TTR structures determined by cryo-EM.

(A) Cryo-EM reconstructions of the (biarylamine-FT₂-WT)₁(C10A)₃ TTR compressed state (determined to ~3.4 Å resolution), colored by subunit. On the right is a cross-sectional view of the cryo-EM density showing the density corresponding to the single bound ligand (green) in the A-C binding pocket, while the B-D binding pocket is empty. (B) The cryo-EM density corresponding to the area denoted by the dashed box in (A) is shown as a mesh with the atomic model shown as a stick model. The pocket volume of each binding site is denoted in green text. (C) Cryo-EM reconstruction of the (biarylamine-FT₂-WT)₁(C10A)₃ TTR compressed frayed state (determined to ~4.1 Å resolution), colored by subunit. On the right is a cross-sectional view of the cryo-EM density showing the density corresponding to the single-bound ligand (green) in the A-C binding pocket, while the B-D binding pocket, which is associated with the less-ordered protomers, is empty.

Figure 4.. Schematic outlining the conformational landscape of TTR and how it is influenced by ligand binding.

A cartoon representation of the TTR tetramer is colored by subunit as in other figures, with the ligand represented as a diamond. In the absence of ligand, the TTR tetramer adopts an equilibrium of conformational states described in this work, with some fraction of the tetramer having a propensity to dissociate into dimers, which could subsequently further dissociate and aggregate. A small molecule ligand can bind one of the two available binding pockets in the TTR tetramer. This binding occurs without incurring a substantial entropic cost, as the ligand binds to the distinct binding pocket that more readily accommodates the interaction. This single ligand binding event is sufficient to increase the stability of the dimer-dimer interactions, lowering the propensity for dimer dissociation and aggregation. The next binding event by the same ligand will incur a much larger entropic cost, as the remaining binding pocket requires structural rearrangements to accommodate the ligand. Furthermore, stabilization of the dimer-dimer interfaces induces an ordering of the loops associated with the frayed state, which comes with an entropic cost.