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

摘要

Amyloid formation of natively folded proteins involves global and/or local unfolding of the native state to form aggregation-prone intermediates. Here we report solid-state NMR structural studies of amyloid derived from wild-type (WT) and more aggressive mutant forms of transthyretin (TTR) to investigate the structural changes associated with effective TTR aggregation. We employed selective ¹³C-labeling schemes to investigate structural features of β-structured core regions in amyloid states of WT and two mutant forms (V30M and L55P) of TTR. Analyses of the ¹³C-¹³C correlation solid-state NMR spectra revealed that WT TTR aggregates contain an amyloid core consisting of native-like CBEF and DAGH β-sheet structures and the mutant TTR amyloids adopt a similar amyloid core structure with native-like CBEF and AGH β-structures. However, the V30M mutant amyloid was shown to have a different DA β-structure. In addition, strand D is more disordered even in the native state of L55P TTR, indicating that the pathogenic mutations affect the DA β-structure, leading to more effective amyloid formation. The NMR results are consistent with our mass spectrometry-based thermodynamic analyses that showed the amyloidogenic precursor states of WT and mutant TTRs adopt folded structures, but the mutant precursor states are less stable than that of WT TTR. Analyses of the oxidation rate of methionine sidechain also revealed that the sidechain of residue Met-30 pointing between strands D and A is not protected from the oxidation in V30M mutant, while protected in the native state, supporting that the DA β-structure might be disrupted in V30M mutant amyloid.