Conformational Flexibility Tunes the Propensity of Transthyretin to Form Fibrils Through Non‐Native Intermediate States

Abstract

The formation of partially unfolded intermediates through conformational excursions out of the native state is the starting point of many diseases involving protein aggregation. Therapeutic strategies often aim to stabilize the native structure and prevent the formation of intermediates that are also cytotoxic in vivo. However, their transient nature and low population makes it difficult to characterize these intermediates. We have probed the backbone dynamics of transthyretin (TTR) over an extended timescale by using NMR spectroscopy and MD simulations. The location and extent of these motions indicates that the backbone flexibility of TTR is a cause of dissociation and destabilization, both of which are responsible for fibril formation. Importantly, approximately 10 % of wild-type TTR exists in an intermediate state, which increased to up to 28 % for pathogenic TTR mutants, for which the formation of the intermediate state is shown to be energetically more favorable compared to the wild type. This result suggests an important role for the intermediates in TTR amyloidosis.

Pathogenic Mutations Induce Partial Structural Changes in the Native β-Sheet Structure of Transthyretin and Accelerate Aggregation

“…However, a notable difference was observed in strand D. The short strand with little flexibility in WT TTR undergoes substantial conformational fluctuations in the pathogenic mutants. 60 These NMR results are largely consistent with our biophysical studies that showed the amyloidogenic states of the TTR variants are less stable than that of WT TTR. In addition, our solid-state NMR results reveal that amyloid states of WT and two mutant forms of TTR adopt a similar native-like CBEF and AGH β-sheet structure, but different DA substructures were observed in the mutant amyloids.…”

“…In addition, previous relaxation dispersion NMR experiments of the monomeric and tetrameric TTR showed that CBEF β-sheet remains stable while the other β-sheet (DAGH) undergoing extensive conformational fluctuations on millisecond time scales associated with misfolding and amyloid formation. 60,63 These NMR results suggest that DAGH β-sheet may be involved in intermolecular associations. Indeed, recent mechanistic studies of TTR aggregation showed that intermolecular interactions between strands H play a key role in TTR amyloid formation, and strand A has a strong aggregation propensity.…”

“…Recent relaxation NMR experiments identified flexible regions undergoing conformational fluctuations that might be linked to the dissociation of the TTR tetramer to aggregation-prone monomers. 60 The pathogenic mutations (V30M and L55P) were shown to induce more extensive conformational fluctuations of the same flexible regions as those observed for WT TTR. However, a notable difference was observed in strand D. The short strand with little flexibility in WT TTR undergoes substantial conformational fluctuations in the pathogenic mutants.…”

“…The longer side chain of methionine than that of valine may crash with other side chains in strands D and A, inducing a structural change in the DA substructure, as was suggested by the previous relaxation dispersion NMR experiments. 60 In the stronger amyloidogenic mutant L55P, the distance between residues P55 and M13 is higher (6.5 Å) even in the native state, in comparison to the native WT tetrameric TTR (5 Å), as shown in Supplementary Figure S5. The strand D is also more disordered in the more amyloidogenic mutant L55P TTR, which is consistent with our mass spectra-based analysis that showed the amyloidogenic state of the mutant is less stable than that of WT TTR.…”

Struktur eines monomeren Transthyretin mit der klinisch wichtigen T119M‐Mutation

“…Relaxationsmessungen des Proteinrückgrats zeigten, dass im Vergleich zu M‐TTR eine geringere Zahl an T119M‐M‐TTR‐Resten 15 N‐Relaxationsdispersion aufweist . Zudem verringert der Einbau der T119M‐Mutation in das Wildtyp‐TTR die Konformationsdynamik innerhalb des tetrameren Zustands . Um darüber hinaus gehende Einblicke in Unterschiede der internen Dynamik zwischen dem monomeren T119M‐M‐TTR und Wildtyp‐TTR zu gewinnen, schätzen wir auf Basis der experimentellen chemischen Verschiebungen und unter Zuhilfenahme des Programms TALOS‐N die Ordnungsparameter des Proteinrückgrats ab.…”