Structural Changes Associated with Transthyretin Misfolding and Amyloid Formation Revealed by Solution and Solid-State NMR

Abstract

Elucidation of structural changes involved in protein misfolding and amyloid formation is crucial for unraveling the molecular basis of amyloid formation. Here we report structural analyses of the amyloidogenic intermediate and insoluble aggregates of transthyretin (TTR) using solution and solid-state NMR spectroscopy. Our solution NMR results show that one of the two main β-sheet structures (CBEF β-sheet) is maintained in the aggregation-competent intermediate, while the other DAGH β-sheet is more flexible on ms time scales. Magic-angle-spinning solid-state NMR revealed that AB loop regions interacting with strand A in DAGH β-sheet undergo conformational changes, leading to the destabilized DAGH β-sheet.

Transthyretin (TTR) is a 127-residue homo-tetrameric protein (55 kDa) that acts a carrier of the thyroid hormone thyroxine and retinol-binding protein. Rate-limiting tetramer dissociation, followed by monomer misfolding and aggregation of wild type (WT) TTR, is known to cause senile systemic amyloidosis (cardiomyopathy).¹ More than 100 single-point mutations accelerate the dissociation and amyloid formation, leading to earlier onset of amyloidoses such as familial amyloidotic polyneuropathy (FAP) and familial amyloidotic cardiomyopathy (FAC).²

The native TTR monomer adopts a primarily β-sheet tertiary structure, in which eight β-strands are arranged into a β-sandwich consisting of two β-sheets comprising strands CBEF and DAGH (Figure 1a).³ The amyloid formation process of the globular protein is initiated by local unfolding to form aggregation-prone intermediates, which self-assemble into amyloid.⁴ It was proposed that aggregation-prone states generated by local unfolding of natively folded proteins retain extensive native-like structures.⁵ Investigating whether there is a native-like structure in amyloidogenic intermediates as well as in amyloid is, therefore, of great importance in understanding the molecular mechanism of misfolding and aggregation.

Figure 1.

(a) A ribbon diagram representation of TTR monomer showing residues (pink) observed in the HSQC spectra. (b) Overlay of ¹H/¹⁵N HSQC NMR spectra of WT TTR (T-TTR) and a monomeric variant (F87M/L110M, M-TTR) acquired at pH 4.4 (15 °C) at a ¹H frequency of 500 MHz. In order to slow down aggregation kinetics, a very low protein concentration of 0.15 mg/mL (12 μM) was used to probe the monomeric aggregation-competent state at pH 4.4, where the aggregation rate reaches a maximum. At higher protein concentrations, no NMR signals were observed at pH 4.4 due to aggregation.

Previous structural studies of TTR suggested that amyloidogenic intermediates and the end product of aggregation, amyloid, contain extensive native-like β-sheet structures. However, the extent of native structure has been controversial.^1,6–9 Previous solid-state NMR structural studies of amyloid formed by a short TTR peptide fragment (105–115) provided valuable structural information on its amyloid state.¹⁰ However, full-length TTR has different amyloidogenic properties and likely forms a distinct amyloid state, requiring structural analyses of the full-length TTR amyloid. In this study, we employed solution and solid-state NMR to scrutinize native-like structural features in amyloidogenic intermediates and to identify critical regions undergoing structural changes during amyloid formation.

TTR dissociation, misfolding and amyloid formation is greatly accelerated under mildly acidic conditions (pH 4 – 5).¹ In order to probe the soluble amyloidogenic intermediate state, a two dimensional ¹H/¹⁵N heteronuclear single-quantum coherence (HSQC) NMR spectrum of WT TTR was acquired at low concentration under the most amyloidogenic pH of 4.4 (Figure 1b). Given that TTR efficiently forms amyloid at higher protein concentrations (> 0.2 mg/ml) under these experimental conditions, the NMR parameters have substantial contributions from amyloidogenic precursor states. It is notable that about one third of the NMR resonances are observable, while the rest of the resonances are undetectable due to broadening from conformational exchange on ms time scales. The remaining NMR resonances are well-dispersed and their chemical shifts are also similar to those of the native tetrameric TTR (BMRB 5507), indicating that the aggregation competent state contains native-like conformations.

The WT tetramer has been shown to substantially dissociate to monomers at acidic pH.¹ Thus, the HSQC spectrum of the WT TTR tetramer (T-TTR) was compared to that of a monomeric TTR variant (F87M/L110M, M-TTR) that exists predominantly as monomer (> 99 % at a protein concentration of 0.1 mM).¹¹ Most of the observable NMR resonances of WT TTR appeared at identical positions with similar linewidth to the cross peaks in the spectrum of M-TTR, suggesting that TTR is predominantly in the monomeric state at the amyloidogenic pH. The observable NMR resonances were assigned using pH-titration experiments of M-TTR¹² and the available backbone assignment of WT tetramer (BMRB 5507; the chemical shifts of the residues in CBEF β-sheet were not affected by the two mutations (F87M/L110M))⁷. Interestingly, the observed NMR resonances in WT TTR are mapped to residues in strands C, B, E and F and the BC loop (Figure 1a), while other resonances are undetectable due to the extensive line-broadening caused by ms time scale dynamics, consistent with our previous relaxation dispersion NMR experiments on M-TTR.¹² The solution NMR results described above indicate that the aggregation-competent state of WT TTR contains native-like structure in the CBEF β-sheet, while the DAGH β-sheet structure appears to be perturbed. In order to investigate structural changes in the DAGH β-sheet, magic-angle-spinning (MAS) solid-state NMR was used for the structural studies of TTR amyloid.

Magic-angle-spinning (MAS) solid-state NMR has provided valuable structural information on various amyloids, such as the location of β-strands and unstructured regions within the amyloid.^13–20 Despite the great progress in solid-state NMR methodology, full structural characterization of the amyloid derived from this 127-residue protein remains an enormous challenge. The current structural study is, therefore, focused on specific regions that may undergo structural changes during amyloid formation (Figure 2a). Previous relaxation dispersion NMR experiments on monomeric variant¹² and tetrameric TTR²¹ demonstrated that AB loop regions undergo extensive conformational exchanges on ms time scales, which might be associated with TTR misfolding and amyloid formation. Thus, six amino acids (A, D, L, R, S, V) were uniformly ¹³C^/15N labeled to achieve sequential labeling for the AB loop regions (Materials and Methods in Supplemental Information).

Figure 2.

(a) A ribbon diagram representation of TTR monomer showing labeled residues (blue) in the AB loop regions. Pro-24 and Leu-17 are labeled in red. (b) Cross-polarization (CP) based two-dimensional ¹³C-¹³C correlation spectra of native (black) and amyloid (red) states of TTR obtained using a mixing time of 50 ms at a ¹H frequency of 800 MHz. 64 FIDs were accumulated for the 2D spectra with a spinning frequency of 11.3 kHz.

Two-dimensional proton-driven spin-diffusion (PDSD)²² experiments were carried out to investigate structural features of the AB loop regions in the native (black) and amyloid (red) states of TTR (Figure 2b and Supplemental Figure S2). The cross-peaks from the AB loop regions are well resolved and unambiguously assigned for the native state based on the selective labeling of the six amino acids and the available backbone assignment (BMRB 5507). The strong cross-peaks in the CP based ¹³C-¹³C correlation spectrum indicate that the AB loop is well structured in the native state. In contrast to the native state, the cross-peaks from the AB loop region (19 – 30) are not observed in amyloid state (Figure 2b).

The solid-state NMR results described above suggest that the loop region may adopt distinct non-native conformations or become disordered in amyloid state. In particular, hydrogen-bonding interactions between the short strand in the AB loop and strand A might be disrupted in amyloid state. In order to examine the structural change, we utilized specific labeling schemes, which generate isolated ¹³C-¹³C dipolar-coupled spin pairs in the native structure (Figure 2a). For example, ¹³CO-Pro and ¹³Cα-Leu labeled protein has two dipolar-coupled ¹³CO-¹³Cα spin pairs at distances of 2–5 Å (P11-L12, 2.4 Å; P24-L17, 4.9 Å). The dipolar-coupled spin pairs in the native tetramer can then be probed and compared to those in amyloid state using solid-state NMR experiments that can detect spin-pairs with a separation of up to 6 Å

2D PDSD²² experiments were carried out to probe the dipolar-coupled spin pairs in the native, and also in the amyloid state(s) (Figure 3a). A longer mixing time of 500 ms was used to probe the weakly-coupled spin pair (P24-L17, 4.9 Å) and the spinning speed was set slightly below the Δω_iso = 2ω_r rotational resonance condition²³ for more efficient polarization transfer. The cross-peaks from the two spin pairs (P11-L12 and P24-L17) were clearly observed in the 2D spectrum of the native tetrameric TTR (the assignment of the cross-peaks were made from the previous assignment, BMRB 5507). On the other hand, only one cross-peak was observed in amyloid state of TTR. In order to assign the cross-peak in the TTR amyloid, ¹³Cα-Val and ¹³Cα-Leu labeled TTR was prepared, resulting in only one V16-L17 (3.3 Å) spin pair with a separation of less than 5 Å. The 2D PDSD experiment on the ¹³Cα-Val and ¹³Cα-Leu labeled TTR confirms the assignment of the cross-peak in the amyloid state (P11-L12, red). The disappearance of the cross-peak from the P24-L17 spin-pair in TTR amyloid supports the occurrence of structural changes in the AB loop regions (Figure 2a). The cross-peak between the two residues may be broadened by conformational disorder of the loop regions and/or the short β-strand in the AB loop and strand A may not be in close proximity in amyloid state.

Figure 3.

(a) 2D PDSD spectra of native (black) and amyloid (red) states of the ¹³CO-Pro and ¹³Cα-Leu labeled TTR. (b) A 2D PDSD spectrum of the amyloid state for the ¹³Cα-Leu and ¹³Cα-Val labeled TTR. The NMR spectra were obtained using a mixing time of 500 ms at a ¹H frequency of 830 MHz.

In order to obtain additional evidence for the conformational disorder of the AB loop regions, the 2D ¹³C-¹³C correlation experiment was carried out on ¹³CO-Pro, ¹³Cα-Ala, and ¹³Cα-Leu labeled TTR amyloid that contains two strongly dipolar-coupled ¹³CO-¹³Cα spin pairs (P11-L12 and P24-A25; 2.4 Å, Supplemental Figure S4). In the 2D PDSD spectrum, the cross-peak for P24-A25 was not detected in amyloid state of TTR, while the cross-peak for P11-L12 was clearly observed in the amyloid state spectra. These NMR results clearly indicate that the AB loop regions are disordered in amyloid state.

Previous solid-state NMR studies of various amyloid states formed by small peptide fragments and full-length proteins (90 – 150 residues) showed that NMR signals are observed mainly from residues in rigid, structured amyloid core regions in the cross-polarization (CP) based solid-state NMR spectra, which utilize substantial dipole-dipole couplings for efficient magnetization transfer from ¹H to ¹³C.^19,20,24,25 NMR peaks are usually not detected from flexible and/or disordered regions due to severe line-broadening and/or inefficient cross-polarization caused by motional averaging of dipolar interactions.^19,20,24,25 The strong cross-peaks observed in TTR amyloid spectra (Figure 2 and 3) are comparable to those of its native tetrameric state, indicating that the amyloid state contains a rigid, structured amyloid core. The presence of a β-structured amyloid core is further evidenced in two-dimensional ¹³C-¹³C correlation MAS spectra (Supplemental Figure S2 and S3). On the other hand, NMR peaks from the AB loop regions in TTR amyloid state were not detectable in the solid-state NMR spectra, suggesting that the loop regions become disordered during the misfolding and amyloid formation.

Recent relaxation dispersion NMR experiments on monomeric variant (F87M/L110M)¹² and tetrameric forms of TTR²¹ revealed that the residues in the DAGH β-sheet and AB loop regions undergo conformational exchange on ms time scales, while residues in the CBEF β-sheet remain stable. The dynamics experiments are in good agreement with our solution and solid-state NMR results. The tetrameric native state of TTR is stabilized by dimerization of dimers through the interactions between the AB and GH loops. The structural perturbations of the AB loop may facilitate dissociations of the native tetramers into monomers, which is the critical step for TTR misfolding and amyloid formation.

In summary, the solution HSQC NMR results described here show that the soluble amyloidogenic precursor state of TTR contains a native-like CBEF β-structure. The stable CBEF β-sheet native structure in the amyloidogenic intermediate state is somewhat contracted to the previous hydrogen/deuterium (H/D) exchange⁶ and protease digestion studies⁹ of TTR amyloids that suggested the CBEF β-structure is partly disrupted in amyloid state. A very recent study, however, showed that TTR amyloid is in a dynamic equilibrium with soluble intermediate states in solution,²⁶ rendering analyses of the H/D exchange and protease digestion studies of TTR amyloid more complicated.

In contrast to the CBEF β-structure, NMR signals from the DAGH β-sheet were not observed in solution due to extensive line-broadening. Our solid-state NMR experiments indicate that the well-structured AB loop regions in the native state become disordered during amyloid formation. The structural perturbations of the AB loop regions including the disruption of the interactions between the short β-strand in the AB loop and strand A may affect the stability of the whole DAGH β-sheet, rendering the β-sheet more dynamic on ms time scales and unobservable in the HSQC NMR spectrum.

ABBREVIATIONS

NMR

nuclear magnetic resonance

HSQC

heteronuclear single-quantum coherence

MAS

magic-angle spinning

CP

cross-polarization

WT

wild-type

TTR

transthyretin

PDSD

proton-driven spin-diffusion

TEM

transmission electron microscopy

ThT

thioflavin T

Leu

leucine

Val

Valine

Pro

proline

H/D

hydrogen/deuterium