A transthyretin monomer intermediate undergoes local unfolding and transient interaction with oligomers in a kinetically concerted aggregation pathway

Sun, Xiyan; Ferguson, James A.; Dyson, H. Jane; Wright, Peter E.

doi:10.1016/j.jbc.2022.102162

Cited by 10 publications

(10 citation statements)

References 53 publications

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“…All of these residues exhibit relaxation dispersion at pH 6.5 (Table S1, Figure S8) and the intensity loss is consistent with enhanced conformational fluctuations at pH 4.4 that lead to exchange broadening. 40 In contrast, amide resonances of residues in the EF helix and strand H become more intense, indicating that these regions become more flexible at acidic pH, suggesting local unfolding. Further analysis of the titration data reveals that the A120 and V121 cross peaks in state B lose intensity as the pH is reduced from 6.7 to 4.4, with concomitant increase in the intensity of the corresponding cross peaks in state A (Figure S10).…”

Section: Resultsmentioning

confidence: 99%

“…14 Nature of the F87E excited state Mutant monomeric TTR is stable near neutral pH but aggregates rapidly when partially unfolded at pH 4.4. 13,14,40 We have previously reported pH titrations of F87E, performed at 277K to decrease the rate of aggregation. 40 As the pH is reduced from 6.7 to 4.4, amide cross peaks associated with residues in strand A, the AB loop, strand D, the DE loop, the EF loop, and the C-terminal end of strand F lose more than 60% intensity.…”

Section: F87e Adopts Two Ground State Conformations That Differ In Dy...mentioning

confidence: 99%

“…13,14,40 We have previously reported pH titrations of F87E, performed at 277K to decrease the rate of aggregation. 40 As the pH is reduced from 6.7 to 4.4, amide cross peaks associated with residues in strand A, the AB loop, strand D, the DE loop, the EF loop, and the C-terminal end of strand F lose more than 60% intensity. All of these residues exhibit relaxation dispersion at pH 6.5 (Table S1, Figure S8) and the intensity loss is consistent with enhanced conformational fluctuations at pH 4.4 that lead to exchange broadening.…”

Section: F87e Adopts Two Ground State Conformations That Differ In Dy...mentioning

confidence: 99%

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Characterization of an amyloidogenic intermediate of transthyretin by NMR relaxation dispersion

Leach,

Ferguson,

Morgan

et al. 2024

Preprint

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The aggregation pathway of transthyretin (TTR) proceeds through rate-limiting dissociation of the tetramer and partial misfolding of the monomers, which assemble into amyloid structures through a downhill polymerization mechanism. The structural features of the aggregation-prone monomeric intermediate are poorly understood. Characterization of amyloidogenic intermediates is challenging due to their propensity to aggregate at concentrations necessary for structural studies. NMR relaxation dispersion offers a unique opportunity to characterize these intermediates when they exchange on favorable timescales with NMR-visible ground states. To characterize the structural transitions associated with tetramer dissociation, we have analyzed ground-state chemical shift differences between the native tetramer and an engineered monomer in which the critical F87 side chain is replaced by glutamate. The secondary structure and overall fold of the F87E monomer is similar to that of the tetramer except for β-strand H. This strand populates two conformations, where it is either docked on the protein core or is displaced from the edge of the β-sheet formed by β-strands D, A, G, and H (DAGH β-sheet) and is dynamically disordered. Chemical shift differences derived from analysis of1H/15N single, double and zero quantum relaxation dispersion data provide insights into the structure of a low-lying excited state that exchanges with the ground state of the F87E monomer at a rate of 3800 s-1. Disruption of the subunit interfaces of the TTR tetramer leads to destabilization of edge strands in both β-sheets of the F87E monomer. Conformational fluctuations are propagated through the entire hydrogen bonding network of the DAGH β-sheet, from the inner β-strand H, which forms the strong dimer interface in the TTR tetramer, to outer strand D which is unfolded in TTR fibrils. Fluctuations are also propagated from the AB loop in the weak dimer interface to the EF helix, which undergoes structural remodeling in fibrils. The conformational fluctuations in both regions are enhanced at acidic pH where amyloid formation is most favorable. The relaxation dispersion data provide insights into the conformational dynamics of the amyloidogenic state of monomeric TTR that predispose it for structural remodeling and progression to amyloid fibrils.

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Section: Resultsmentioning

confidence: 99%

Section: F87e Adopts Two Ground State Conformations That Differ In Dy...mentioning

confidence: 99%

Section: F87e Adopts Two Ground State Conformations That Differ In Dy...mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of an amyloidogenic intermediate of transthyretin by NMR relaxation dispersion

Leach,

Ferguson,

Morgan

et al. 2024

Preprint

Self Cite

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“…The rate-limiting step in TTR aggregation is the dissociation of the tetramer to form an aggregation-prone monomeric intermediate. ,,, Dissociation of WT TTR at neutral pH is on the order of days, and previous in vitro studies of the TTR aggregation pathway were therefore performed at acidic pH to accelerate dissociation of the tetramer and promote local unfolding and aggregation of the resulting monomer. ,,,, Although tetramer dissociation at neutral pH has been observed for WT TTR at sub-μM concentration and at μM concentration for the highly destabilized variants L55P, , V14D-V16E, S112I, , and D18G, the thermodynamic equilibrium constants of individual steps, the molecular nature of dissociative intermediates, and the kinetics of the dissociation pathway are unknown. Understanding the kinetics and mechanism of TTR tetramer dissociation and reassembly at neutral pH is critical for understanding transthyretin amyloidosis under physiological conditions.…”

Section: Discussionmentioning

confidence: 99%

Probing the Dissociation Pathway of a Kinetically Labile Transthyretin Mutant

Sun,

Ferguson,

Leach

et al. 2023

J. Am. Chem. Soc.

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Aggregation of transthyretin (TTR) is associated with devastating amyloid diseases. Amyloidosis begins with the dissociation of the native homotetramer (a dimer of dimers) to form a monomeric intermediate that assembles into pathogenic aggregates. This process is accelerated in vitro at low pH, but the process by which TTR dissociates and reassembles at neutral pH remains poorly characterized due to the low population of intermediates. Here, we use 19 F-nuclear magnetic resonance (NMR) and a highly sensitive trifluoromethyl probe to determine the relative populations of the species formed by the dissociation of a destabilized variant, A25T. The A25T mutation perturbs both the strong dimer and weak dimer−dimer interfaces. A tetramer ⇌ dimer ⇌ monomer (TDM) equilibrium model is proposed to account for concentration-and temperature-dependent population changes. Thermodynamic and kinetic parameters and activation energetics for dissociation of the native A25T tetramer, as well as a destabilized alternative tetramer (T*) with a mispacked F87 side chain, were extracted by van't Hoff and 19 F-NMR line shape analysis, saturation transfer, and transition state theory. Chemical shifts for the dimer and T* species are degenerate for 19 F and methyl probes close to the strong dimer interface, implicating interfacial perturbation as a common structural feature of these destabilized species. All-atom molecular dynamics simulations further suggest more frequent F87 ring flipping on the nanosecond time scale in the A25T dimer than in the native A25T tetramer. Our integrated approach offers quantitative insights into the energy landscape of the dissociation pathway of TTR at neutral pH.

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“…Additionally, these methods can be applied to studying the initial molecular events associated with neurodegenerative diseases that involve oligomers either in the pathology or as a prenucleation step in a fibril aggregation process. For systems like Aβ 40 , 37 Aβ 42 , 38 Huntington, 39 and transthyretin, 40 these techniques may enhance the understanding of how the proteins misfold and form toxic aggregates, potentially a key step in developing therapies to slow or stop progression of such diseases.…”

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confidence: 99%

From Milliseconds to Minutes: Melittin Self-Assembly from Concerted Non-Equilibrium Pressure-Jump and Equilibrium Relaxation Nuclear Magnetic Resonance

Gelenter,

Yau,

Anfinrud

et al. 2024

J. Phys. Chem. Lett.

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Non-equilibrium kinetics techniques like pressure-jump nuclear magnetic resonance (NMR) are powerful in tracking changes in oligomeric populations and are not limited by relaxation rates for the time scales of exchange that can be probed. However, these techniques are less sensitive to minor, transient populations than are Carr−Purcell−Meiboom− Gill (CPMG) relaxation dispersion experiments. We integrated non-equilibrium pressure-jump and equilibrium CPMG relaxation dispersion data to fully map the kinetic landscape of melittin tetramerization. While monomeric peptides weakly form dimers (K d,D/M ≈ 26 mM) whose population never exceeds 1.6% at 288 K, dimers associate tightly to form stable tetrameric species (K d,T/D ≈ 740 nM). Exchange between the monomer and dimer, along with exchange between the dimer and tetramer, occurs on the millisecond time scale. The NMR approach developed herein can be readily applied to studying the folding and misfolding of a wide range of oligomeric assemblies.

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A transthyretin monomer intermediate undergoes local unfolding and transient interaction with oligomers in a kinetically concerted aggregation pathway

Cited by 10 publications

References 53 publications

Characterization of an amyloidogenic intermediate of transthyretin by NMR relaxation dispersion

Characterization of an amyloidogenic intermediate of transthyretin by NMR relaxation dispersion

Probing the Dissociation Pathway of a Kinetically Labile Transthyretin Mutant

From Milliseconds to Minutes: Melittin Self-Assembly from Concerted Non-Equilibrium Pressure-Jump and Equilibrium Relaxation Nuclear Magnetic Resonance

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