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

Das, Jitendra K.; Mall, Shyam S.; Bej, Aritra; Mukherjee, Sujoy

doi:10.1002/ange.201407323

Cited by 7 publications

(10 citation statements)

References 27 publications

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“…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 nativelike CBEF and AGH β-sheet structure, but different DA substructures were observed in the mutant amyloids.…”

Section: ■ Discussionsupporting

confidence: 90%

“…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.…”

Section: ■ Discussionmentioning

confidence: 78%

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

Lim

Dasari

et al. 2017

Biochemistry

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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.

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Section: ■ Discussionsupporting

confidence: 90%

Section: ■ Discussionmentioning

confidence: 78%

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

Lim

Dasari

et al. 2017

Biochemistry

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“…A previous report of 15 N relaxation dispersion experiments on WT TTR and the V30M and L55P mutants is in qualitative agreement with our current results, revealing structural fluctuations in the F, G, and H strands and the AB loop that mediate the propensity to form fibrils. 46 However, the published data exhibit considerable variation in the location of the dispersing residues and in the magnitude of the exchange contributions (R ex ) for the different TTR variants, similar to the results we obtained in preliminary experiments when expression and purification protocols were not sufficiently well-controlled. Despite differences in pH, the reported exchange rates (k ex ) are the same as ours within experimental uncertainties but the excited state populations are higher.…”

Section: ■ Discussionsupporting

confidence: 83%

“…Despite differences in pH, the reported exchange rates (k ex ) are the same as ours within experimental uncertainties but the excited state populations are higher. 46 In addition, data for the V30M and L55P mutants were acquired at a different temperature from wild-type, making quantitative comparison difficult. As detailed in the experimental section, great care was taken in the present work to produce carefully matched TTR samples and acquire relaxation dispersion data under identical solution conditions, which likely accounts for the differences between the previous results and those reported here.…”

Section: ■ Discussionmentioning

confidence: 99%

NMR Measurements Reveal the Structural Basis of Transthyretin Destabilization by Pathogenic Mutations

et al. 2018

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Inherited mutations of transthyretin (TTR) destabilize its structure, leading to aggregation and familial amyloid disease. Although numerous crystal structures of wild-type (WT) and mutant TTRs have been determined, they have failed to yield a comprehensive structural explanation for destabilization by pathogenic mutations. To identify structural and dynamic variations that are not readily observed in the crystal structures, we used NMR to study WT TTR and three kinetically and/or thermodynamically destabilized pathogenic variants (V30M, L55P, and V122I). Sequence-corrected chemical shifts reveal important structural differences between WT and mutant TTR. The L55P mutation linked to aggressive early onset cardiomyopathy and polyneuropathy induces substantial structural perturbations in both the DAGH and CBEF β-sheets, whereas the V30M polyneuropathy-linked substitution perturbs primarily the CBEF sheet. In both variants, the structural perturbations propagate across the entire width of the β-sheets from the site of mutation. Structural changes caused by the V122I cardiomyopathy-associated mutation are restricted to the immediate vicinity of the mutation site, directly perturbing the subunit interfaces. NMR relaxation dispersion measurements show that WT TTR and the three pathogenic variants undergo millisecond time scale conformational fluctuations to populate a common excited state with an altered structure in the subunit interfaces. The excited state is most highly populated in L55P. The combined application of chemical shift analysis and relaxation dispersion to these pathogenic variants reveals differences in ground state structure and in the population of a transient excited state that potentially facilitates tetramer dissociation, providing new insights into the molecular mechanism by which mutations promote TTR amyloidosis.

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“…We also observed increased dynamics in several loops, such as the AB loop, which is structured under physiological conditions but disordered under amyloidogenic conditions. 81 On the basis of our results, the loss of antiaggregation safeguards facilitates conformational changes and allows solvent molecules to participate in and drive secondary structure changes in TTR. This suggests that water, which is already known to aid in protein folding 82 and unfolding, 83 can also actively contribute to protein misfolding.…”

Section: Biochemistrymentioning

confidence: 72%

Drivers of α-Sheet Formation in Transthyretin under Amyloidogenic Conditions

Childers

Daggett

2019

Biochemistry

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Amyloid diseases make up a set of fatal disorders in which proteins aggregate to form fibrils that deposit in tissues throughout the body. Amyloid-associated diseases are challenging to study because amyloid formation occurs on time scales that span several orders of magnitude and involve heterogeneous, interconverting protein conformations. The development of more effective technologies to diagnose and treat amyloid disease requires both a map of the conformations sampled during amyloidogenesis and an understanding of the molecular mechanisms that drive this process. In prior molecular dynamics simulations of amyloid proteins, we observed the formation of a nonstandard type of secondary structure, called α-sheet, that we proposed is associated with the pathogenic conformers in amyloid disease, the soluble oligomers. However, the detailed molecular interactions that drive the conversion to α-sheet remain elusive. Here we use molecular dynamics simulations to interrogate a critical event in transthyretin aggregation, the formation of aggregation-competent, monomeric species. We show that conformational changes in one of the two β-sheets in transthyretin enable solvent molecules and polar side chains to form electrostatic interactions with main-chain peptide groups to facilitate and modulate conversion to α-sheet secondary structure. Our results shed light on the early conformational changes that drive transthyretin toward the α-sheet structure associated with toxicity. Delineation of the molecular events that lead to aggregation at atomic resolution can aid strategies to target the early, critical toxic soluble oligomers.

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

Cited by 7 publications

References 27 publications

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

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

NMR Measurements Reveal the Structural Basis of Transthyretin Destabilization by Pathogenic Mutations

Drivers of α-Sheet Formation in Transthyretin under Amyloidogenic Conditions

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