Localized Structural Fluctuations Promote Amyloidogenic Conformations in Transthyretin

Abstract

The process of transthyretin (TTR) misfolding and aggregation, including amyloid formation, appears to cause a number of degenerative diseases. During amyloid formation, the native protein undergoes a tetramer-to-folded monomer transition, followed by local unfolding of the monomer to an assembly-competent amyloidogenic intermediate. Here we use NMR relaxation dispersion to probe conformational exchange at physiological pH between native monomeric transthyretin (the F87M/L110M variant) and a small population of a transiently formed amyloidogenic intermediate. The dispersion experiments show that a majority of the residues in the β-sheet containing β-strands D, A, G and H undergo conformational fluctuations on μs-ms time scales. Exchange broadening is greatest for residues in the outer β-strand H, which hydrogen bonds to β-strand H’ of a neighboring subunit in the tetramer, but the associated structural fluctuations propagate across the entire β-sheet. Fluctuations in the other β-sheet are limited to the outer β-strand F, which packs against strand F’ in the tetramer, while the B, C, and E β-strands of this sheet remain stable. The structural changes were also investigated under more forcing amyloidogenic conditions (pH 6.4–3.7), where β-strand D and regions of the D-E and E-F loops were additionally destabilized, increasing the population of the amyloidogenic intermediate and accelerating amyloid formation. Strands B, C, and E appear to maintain native-like conformations in the partially unfolded, amyloidogenic state of wild type TTR. In the case of the protective mutant T119M, the conformational fluctuations are suppressed under both physiological and mildly acidic conditions, indicating that the dynamic properties of TTR correlate well with its aggregation propensity.

NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers

“…Dissociation of the tetrameric two-sheet β-sandwich TTR into a monomeric form and subsequent misfolding of the monomer has been postulated as the mechanism leading to its amyloid fibril formation (74). A monomeric variant of TTR has now been shown by RD NMR to transiently populate an intermediate state characterized by structural fluctuations in one of the sheets, suggesting that amyloid formation in this case may occur by partial rather than global unfolding (75). Transiently populated states have also been observed in the cataract-causing Opj variant of γS-crystallin, a structural protein in the eye lens (76).…”

Relaxation Dispersion NMR Spectroscopy

“…Relaxation dispersion experiments cannot only be used to learn how proteins fold into their native structure, but also to derive mechanisms that underlie protein misfolding and aggregation (Farber et al 2012;Lim et al 2013;Neudecker et al 2012). Since protein misfolding is a characteristic of several neurodegenerative diseases, such as Alzheimer's disease, it is of fundamental importance to understand its mechanism on a molecular level to allow for the development of more efficient treatments.…”

“…An immediate disease related protein misfolding study was conducted on transthyretin (TTR) whose amyloid formation is linked to several degenerative diseases such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy (Lim et al 2013). TTR is a homo-tetrameric protein whose monomers exhibit a β-sandwich structure and it is known that its amyloid formation involves a transition from the tetramer via a folded monomer into an assembly-prone amylogenic intermediate:…”

Using Molecular Dynamics Simulations as an Aid in the Prediction of Domain Swapping of Computationally Designed Protein Variants

“…Protein variants with altered dynamics may lead to undesired outcomes such as amyloidogenesis [13,14]. The goal of many protein engineering projects is therefore to maintain the basic structure and dynamics of the protein while improving a desired property (e.g., catalytic activity [15], thermostability [16], substrate specificity [17], ligand binding [18], and molecular transport [19]).…”