Crystal Structures of Human Transthyretin Complexed with Glabridin

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Crystal Structures of Human Transthyretin Complexed with Glabridin Takeshi Yokoyama, Yuto Kosaka, and Mineyuki Mizuguchi* Faculty of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0914, Japan S Supporting Information *

ABSTRACT: Transthyretin (TTR) is a plasma protein implicated in human amyloid diseases. Several small molecules that bind to the thyroxine-binding site of TTR have been shown to stabilize the TTR tetramer and to inhibit amyloid fibril formation of TTR. Herein, we demonstrated that glabridin (Glab), a prenylated isoflavan isolated from Glycyrrhiza glabra L., inhibited aggregation of TTR in a thioflavin assay. The TTR−Glab complex structure revealed a novel binding mode including a CH−π interaction with A108 and a hydrogen bond with K15. A structural comparison with the wild type-apo structure revealed that the CH−π interaction with A108 was strengthened by the induced-fit conformational change upon Glab binding. Furthermore, the binding of Glab induced a rotation of the T119 side chain, and the inclusion of a water molecule, leading to stabilization of the dimer−dimer interface. These results demonstrate that Glab is a novel inhibitor of TTR fibrillization and suggest the molecular mechanism by which Glab binding stabilizes the tetramer.

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INTRODUCTION Amyloid fibrils are formed by denaturation and misfolding of amyloidogenic proteins, such as amyloid-β peptide, prion,1 and transthyretin (TTR).2 TTR amyloid fibrils are found in patients afflicted with familial amyloidotic polyneuropathy (FAP),3 familial amyloid cardiomyopathy (FAC),4 and senile systemic amyloidosis (SSA).5 To date, more than 100 mutations, including the V30M mutation, have been shown to induce amyloidoses such as FAP and FAC.6,7 TTR is found in human plasma, where it binds to retinolbinding protein and to thyroxine (T4). 8 TTR is a homotetrameric β-sheet-rich protein composed of four subunits, termed A, B, C and D. Each subunit is composed of 127 amino acid residues and has eight β-strands designated A−H and a short α-helix termed EF-helix. The TTR tetramer is formed by the association of two dimers (the AB dimer and CD dimer). The dimer−dimer contacts predominantly involve several hydrogen bonds and hydrophobic interactions between residues located in the AB and GH loops. The TTR tetramer contains two funnel-shaped T4-binding sites, each defined by a dimer−dimer interface. Under certain conditions, TTR can unfold and aggregate into amyloid fibrils.9 The dissociation of the TTR tetramer into the monomer appears to be the ratelimiting step for the amyloid fibril formation.10 The stabilization of the TTR tetramer by small molecules, which bind to the two T4-binding sites, is a promising strategy for stalling the amyloidogenic potential of TTR.11 This binding is known to stabilize the normally folded tetramer of TTR, thus preventing the conformational changes required for amyloidogenecity.12 It is well-recognized that consumption of fruits and vegetables can reduce the incidence of degenerative diseases, including cancer, heart disease, inflammation, and arthritis.13,14 These protective effects are considered mainly to be due to the presence of various antioxidants. Glabridin (Glab; Figure 1) is a © 2014 American Chemical Society

Figure 1. Structures of Glab, Equo, Daid, and Lute.

prenylated isoflavane originally isolated from the roots of Glycyrrhiza glabra L. (Favaceae), commonly known as licorice, which is used in cosmetics, food, and tobacco and in both traditional and herbal medicine. Glab is considered to be a phytoestrogen and is associated with numerous biological properties, including antioxidant, anti-inflammatory, neuroprotective, antitumorigenic, and skin-whitening activities.15 Since its first isolation and characterization, the number of publications dealing with its chemical and biological characterization has increased exponentially.15 Previously, several small molecules, such as diflunisal, tafamidis, and natural flavonoids, have been shown to bind to the T4-binding site and inhibit the amyloid fibril formation of Received: November 27, 2013 Published: January 14, 2014 1090

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TTR.16−18 There is also some evidence that the prenylated compounds bind to TTR.19,20 However, a crystal structure complexed with a prenylated compound such as Glab has not been reported yet. Because prenylation contributes strongly to the diversification of flavonoids, resulting in an abundance of 1000 prenylated flavonoids in plants,21 an investigation into the molecular interactions between TTR and Glab will be a good starting point for the development of amyloid fibril inhibitors. In the present study, we used a thioflavin T assay to investigate whether Glab inhibits TTR amyloid fibril formation. Our data revealed that Glab was a potent amyloid fibril formation inhibitor with an inhibitory activity equal to that of diflunisal, which is one of the most potent inhibitors. Furthermore, we determined the X-ray crystal structures of TTR complexed with Glab. The present structures reveal a characteristic binding mode of Glab and the molecular mechanisms of the structural stabilization by binding of Glab.

resolution, respectively (SI Table 1). All crystals belonged to space group P21212, and were isomorphous with typical TTR crystal structures deposited in the Protein Data Bank. There were two monomers (subunits A and B) in an asymmetric unit forming a tetramer by crystallographic symmetry operation (Figure 2a). The subunit A was crystallographically identical to the subunit C; the subunit B, to the subunit D. Glab was found to bind to the T4-binding sites located on the 2-fold symmetry axis between the subunits A and C and between the subunits B and D. The omit difference Fourier maps and composite omit Fourier maps clearly elucidated the Glab-binding modes (Figure 2b, c). Because the 2-fold axis crosses the two T4binding sites exactly in the middle, the two symmetry-related Glab molecules were refined with 50% occupancy. The two symmetry-related Glab molecules were well ordered and refined to B factors higher than the protein atoms, but consistent with 50% occupancy (Figure 2b, c; SI Table 1). The root-mean-square deviations (rmsd) of Cα atoms between the WT-apo structure and WT-Glab complex tetramer was 0.24 Å, suggesting that the global folding of the WT-Glab complex was similar to that of WT-apo. Similarly, the rmsd value of Cα atoms between the WT-apo and V30M−Glab tetramer was 0.34 Å, suggesting that the Glab binding to V30M also did not induce any significant structural change. Binding Modes of Glab. Although Glab was found to bind to the T4-binding site in a manner similar to other flavonoids, the orientation was opposite that of these other binding modes. The B-ring of Glab turned to the outside of TTR, whereas the B-ring of luteorin (Lute) turned to the inside of TTR (Figures 1, 3). The other flavonoids are known to bind in the same direction as Lute.18 Taking the hydrophobic cyclized prenyl group into consideration, this binding direction of Glab seemed to be energetically reasonable: the cyclized prenyl group interacts with the hydrophobic pocket in the T4-binding site, and the polar B-ring is solvent-exposed. The interfaces between Glab and TTR were mainly composed of hydrophobic contacts associated with L17, A108, and L110 (Figure 4). A108-Cβ was 3.8 Å from the center of the aromatic A-ring, indicating a CH−π interaction. The only hydrogen bond was found between 2′-OH and K15-Nζ. These two specific interactions seemed to dictate the binding mode of Glab. The 4′-OH of the B-ring cannot form specific hydrogen bonds because it was completely outside the TTR molecule. Although 1-O and 1″-O can be acceptors of a hydrogen bond, there are no hydrophilic interactions involving 1-O and 1″-O in the complex. L110(C)Cβ was 3.6 Å distant from 1″-O, and L17(A)-Cδ1 was 3.6 Å distant from 1-O. These distances are almost the same as the van der Waals distances so that the existence of a CH···O hydrogen bond could not be affirmed. The binding modes and Glab recognition of V30M were similar to those of WT because the rmsd values of Glab molecules (24 atoms) between WTGlab and V30M−Glab were 0.31 (AC site) and 0.34 (BD site) Å. Structural Stabilization by Glab Binding. To elucidate the molecular mechanism of structural stabilization by the binding of Glab, the structure of WT-Glab was analyzed in detail by comparison with the WT-apo structure. In this paper, subunit A was used as the standard subunit for comparison, since our focus was on the quaternary structure. It should be noted again that subunits A and C or subunits B and D are crystallographically identical. Subunit A of WT-Glab was superimposed on that of WT-apo, and the rmsd of each amino acid residue was plotted (Figure 5a). Four large

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RESULTS Inhibitory Potency of Glab. The V30M mutated TTR, the most common TTR variant in FAP patients, was used for the acid-mediated aggregation experiments. The amyloid fibril inhibitory activities of Glab, (R)-equol (Equo), daidzein (Daid), genistein (Geni), luteorin (Lute), and diflunisal (Difl) were investigated against 10 μM V30M TTR (tetramer concentration) using a thioflavin T assay which quantifies the levels of misfolded protein. Daid is an isoflavone that is considered to be a precursor of Glab (Figure 1).15 Although it is not known whether Glab and Equo inhibit TTR amyloid fibril formation, Daid, Geni, Lute, and Difl are known as inhibitors.15,16,22 The inhibitory data of these compounds were collected for comparison. The amyloid fibril inhibitory ratio at each concentration of each compound was plotted, and the EC50 values were calculated by fitting the data to the 4Parameter Logistic Model23 (Supporting Information (SI) Figure 1, Table 1). The thioflavin T assays revealed that the Table 1. Activities of Glab and Selected Compounds glabridin (R)-equol daidzein genistein luteorin diflunisal

EC50 (μM)

SD

r2

6.4 13 16 7.8 6.4 6.3

0.39 0.52 0.60 0.072 0.60 0.23

0.981 0.994 0.998 0.994 0.996 0.996

These values were calculated from triplicate experiments.

EC50 value of Glab was 6.4 μM, the highest potency observed for all three compounds (Glab, Equo, and Daid) against V30M TTR. In the comparison with Difl, which is considered a candidate for clinical use,24−26 we found that the potency of Glab was similar to that of Difl. The inhibitory potency of Equo (13.0 μM) was lower than that of Glab, suggesting that the 2′OH and prenyl group of Glab were important to stabilize the TTR structure. On the other hand, the addition of a carbonyl group to the C-ring and the saturation of the C-ring did not significantly change the inhibitory potency, considering the EC50 values of Equo and Daid (15.7 μM). These results suggested that the high potency of Glab was mainly due to the 2′-OH and cyclized prenyl group. Overall Structure. The crystal structures of WT-apo, WTGlab, and V30M−Glab were solved at 1.6, 2.0, and 1.8 Å 1091

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Figure 2. (a) The quaternary structure of TTR bound to Glab. TTR is represented using a cartoon model, and Glab molecules are represented with a stick model. Subunit A is green, B is yellow, C is cyan, and D is magenta. (b) Omit difference Fourier map around Glab (WT-Glab, |Fo| − |Fc|, 2.0 σ). The Glab molecule and its symmetry-related molecule are shown with a ball-and-stick model. (c) Omit difference Fourier map around Glab (V30M−Glab, |Fo| − |Fc|, 3.3 σ).

Figure 3. Comparison of the binding modes between Glab and Lute. The WT-Lute structure (PDB ID: 4DEW) was superimposed on the WT-Glab structure. Ligands are represented using a stick model. The carbon atoms of Glab are silver; those of Lute are blue. Proteins are represented using a ribbon model. Subunit A of WT-Glab is green, and B is yellow, as in Figure 2. The ribbon model of WT-Lute is blue.

Figure 4. Structure of the Glab-binding site (AC site) of WT-Glab. Carbon atoms of Glab are silver; those of subunit A, green; those of C, cyan; oxygen atoms, red; nitrogen atoms, blue. The hydrogen bond is indicated as a dashed yellow line. 2|Fo| − |Fc| electron density maps are contoured at 1.1σ.

deviations were found at R21/G22, E92, A108, and T119. Similar structural deviations were found between V30M−Glab and WT-apo (Figure 5b). It was expected that the deviations at V14 and M30 were induced by the mutation of V30M (SI Figure 2). The side chain of V14 faces the side chain of V30 (M30), forming hydrophobic interactions. In this way, the mutation to the bulky methionine induced the structural deviation of V14. The deviations found at E92 and A108 were closely binding. The position of A108(C) in WT-Glab was closer to Glab than that in WT-apo, and A108(C)-Cβ in WT-Glab was 3.8 Å distant from the aromatic A-ring, forming a CH−π interaction (Figure 6). This proximity of A108 stabilized the CH−π

interaction. On the other hand, E92 was located on the F strand (residues 91−97), which binds tightly to the G strand (residues 104−112) with several hydrophobic contacts, and thus, the large deviation found at E92 would be due to the movement of A108. The deviations at R21/G22 and T119 were also caused by the Glab binding. The side chain of T119(C) rotated upon Glab binding (Figure 6). Because the 2″-CH3 of Glab was 4.3 Å from T119(C)-Cγ2, the hydrophobic interaction of T119(C)1092

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of subunit D (GH loop: residues 112−115), and both loops are located at the dimer−dimer interface, which is known to be important for the TTR tetramer stability.27 The movement of the Y114(D) side chain was observed upon the Glab binding (Figure 6). The side chain of Y114(D) is known to be involved in several CH···O hydrogen bonds with the AB loop of subunit A.28 It is likely that the structural change in the AB loop induces the movement of Y114(D). The substitution of Y114 with histidine, which is known to be an amyloidogenic variant, perturbed the stability of the quaternary structure.29 Because the Y114 side chain is associated with the quaternary stability of TTR, as mentioned above, we concluded that the observed structural change of the Y114 side chain influences the quaternary stability. Taken together, these results show that the binding of Glab induced the conformational change of T119(C) and the introduction of a water molecule (Figure 6). This conformational change caused the structural change in the AB loop of subunit A and stabilized it by forming the hydrogen-bond network, and the movement of the Y114(D) side chain located at the GH-loop was sequentially induced. These structural changes stabilize the quaternary structure by strengthening the dimer−dimer interactions between the AB-loop and GH-loop. The rmsd plots clearly reflected these conformational changes (Figure 5a, b).

Figure 5. The root-mean-square deviations of all the Cα atoms referred to WT-apo for subunit A. N-terminal residues (1−10) and Cterminal residues (125−127) are excluded because they are disordered. (a) WT-Glab complex. 0 σ (average, 0.18 Å) and 2.5 σ (0.41 Å) are indicated as dashed lines (SD = 0.09 Å). Large deviations are clearly seen at R21/G22, E92, A108, and T119. (b) V30M−Glab complex. 0 σ (average, 0.21 Å) and 2.5 σ (0.55 Å) are indicated as dashed lines (SD = 0.14 Å).

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Cγ2 was favored over the polar T119(C)-Oγ1. As a result of the rotation of T119, one water molecule entered into the binding pocket and formed a hydrogen-bond network (Figure 6). This water molecule formed hydrogen bonds with the carbonyl group of D18(A), the T119(C)-Oγ1, and the carbonyl group of A120(C). The subunit interface between A and C is stabilized not only by the Glab binding but also by the hydrogen-bond network involving this water molecule. Furthermore, the carbonyl group of A120(C) formed a CH···O hydrogen bond with G22(A)-Cα (3.2 Å), and the carbonyl group of D18(A) formed a hydrogen bond with the amino group of G22(A) (3.0 Å). Consequently, this water molecule probably also stabilizes the AB loop region (residues 19−28). The structural deviation observed at R21/G22 was likely to be due to this hydrogenbond network. The above-described structural changes occurring upon Glab binding also contributed to the stabilization of the quaternary structure. The AB loop of subunit A interacts with the GH loop

DISCUSSION In the search for new drugs, it is important to understand the molecular mechanisms underlying the ligand recognition of proteins. Several models, such as a lock and key, induced-fit, and pre-existing equilibrium model, have been proposed so far.30,31 The lock and key model is suitable for explaining the TTR−ligand relationship because the structural changes occurring upon ligand binding (with the exception of the rotations of a few amino acid side chains) have not been observed or discussed in the past research. However, the structural change of A108 observed in the TTR−Glab structure was obviously due to an induced fit, and thus, the main chain of TTR was adapted to the molecular shape of the ligand. We compared the three-dimensional structure of Glab with other inhibitors, which provided an explanation as to why an induced fit was distinctively observed in the TTR−Glab complex. A recent study investigated the protein-binding properties of

Figure 6. Structures of WT-Glab and WT-apo around the Glab-binding site in a wall-eyed stereo representation. The subunit A of WT-Glab was superimposed on that of WT-apo. Carbon atoms of Glab are silver; those of subunit A, green; those of C, cyan; those of D, magenta; oxygen atoms, red; and nitrogen atoms, blue. The carbon atoms of WT-apo are peach. Yellow dashed lines indicate hydrogen bonds, CH−π interactions, or CH···O hydrogen bonds. A water molecule is shown as a sphere with an electron density map (2|Fo| − |Fc|, contoured at 1.3 σ). 1093

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and by the induced fit observed around A108. The binding of Glab stabilized the dimer−dimer interface via the structural change of T119 and the water-mediated hydrogen bond network. The information presented here will be valuable for the design of new amyloidosis inhibitors with improved hydrophobic interactions.

compounds from natural and synthetic sources and found that protein-binding selectivity correlates with shape complexity (defined as the relative content of sp3-hybridized carbons) and stereochemical complexity (defined as the relative content of stereogenic carbons).32,33 The uneven molecular surface of Glab seems to fit to the T4-binding site because of the sp3hybridized carbons of Glab. Although past research has suggested that a hydrogen bond with S117 was important for the ligand recognition of TTR,16,18 no hydrogen bond, including S117 or T119, was found in the TTR−Glab complex. Thus, the inhibitory potency of Glab (EC50 = 6.4 μM) could be accounted for by the relatively large hydrophobic surface of Glab. Hydrophobic interactions by L17, A108, and L110 have also been observed in the other TTR−ligand complexes. The present study indicates that the inhibitory potency could be accounted for not only by the simple calculation of the number of hydrogen bonds, but also by examining how these residues interact with the ligand. Hydrophobic interactions are obviously beneficial to the binding energy as a result of the absence of desolvation energy. The development of TTR amyloid fibril inhibitors will thus be promoted by taking hydrophobic interactions into account, along with CH···O hydrogen bond, CH−π interaction. In recent years, we have determined the neutron crystal structure of TTR,28 but have not yet determined the neutron crystal structure of the TTR−ligand complex. Neutron protein crystallography will allow quantitative analysis of the hydrophobic interaction energy because it will allow us to determine the configuration of the methyl groups.34 The conformational change of T119 and the water molecule introduction observed in TTR−Glab agree with the previously reported structure and MD simulations, which showed the conformational changes upon binding of T4.35 We additionally proposed that this water molecule plays an important role in stabilizing the quaternary structure of TTR. The water molecule stabilized the AB loop by forming a hydrogen-bond network, and these structural changes subsequently induced the conformational change of the Y114 side chain, which is crucial for the stability of the TTR quaternary structure. From the recent crystallographic analysis of the I84S mutant TTR in complex with an inhibitor, it was proposed that the effect of the ligand is not direct.36 Because I84S is 20 Å apart from the T4binding site, the region containing I84T is not directly affected by the ligands. However, the EF loop (residues 82−90) including I84S is close to Y114 so that the stabilization of Y114 would result in stabilization of the EF loop. The structural change of Y114 observed in the present study probably contributes to the stabilization of the EF loop. We conclude that the stabilization of the TTR tetramer results from these sequential structural changes upon the Glab binding, leading to the inhibition of amyloid fibril formation. This stabilization mechanism could also account for the stabilization of the TTR tetramer by a single ligand-binding event37 because the Glab binding to the AC site affected the conformational change of Y114(D). In conclusion, the crystal structure of the TTR−Glab complex provided a picture of the novel binding mode of Glab and led us to propose a water-mediated quaternary stabilization mechanism. Thioflavin T assays revealed that Glab was an inhibitor with the same degree of inhibitory activity as Difl and Lute. Unlike Lute, Glab bound to TTR with several hydrophobic contacts, including a CH−π interaction of A108. The potency of Glab could be explained by its uneven surface

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EXPERIMENTAL PROCEDURES

Materials. Both the wild type TTR (WT) and V30M mutated TTR (V30M) were prepared from an Escherichia coli expression system, as previously described.38 Glab was purchased from Wako Pure Chemical Industries, Ltd. (R)-Equol, daidzein, and genistein were purchased from Cayman Chemical Company. All chemicals were supplied in >95.0% purity as determined by HPLC (Glab, 97%; Equo, 98%; Geni, 98%; Daid, 95%). Acid-Mediated Aggregation Experiments (Thioflavine T Assay). The thioflavin T assay was performed as follows: Ten millimolar stock solutions of Glab, Equo, Daid, Geni, Lute, and Difl were prepared by dissolving the compounds in ethanol. Prior to acidmediated aggregation, 50 μL of V30M (100 μM tetramer concentration) was incubated for 30 min in the presence of compounds at room temperature. After the incubation, the pH was decreased to 4.5 by adding 450 μL of 50 mM sodium acetate, pH 4.5. The sample was gently vortexed and incubated for 96 h at 310 K. At this time, the concentrations of V30M and the compounds were 10 μM and 2.5−40 μM, respectively. For the fluorescence measurements, the incubated solutions at pH 4.5 were 5-fold-diluted with 200 mM Tris−HCl (pH 8.0) in the presence of 20 μM thioflavin T. Fluorescence emission spectra were obtained with excitation and emission wavelengths of 440 and 484 nm, respectively. 100% control inhibition was obtained from the sample at pH 8.0 without decreasing the pH; 0% control inhibition (or baseline) was obtained from the sample in the absence of ligands. The amyloid fibril inhibitory ratio (percent) was calculated as follows: Amyloid fibril inhibitory ratio (%) = 100 × [1 − (fluorescence at each concentration of the compounds − 100% control)/(0% control − 100% control)]. Amyloid fibril inhibitory curves were fitted using the 4-parameter logistic model23 by the least-squares methods, and at least five ligand concentrations were used. EC50 was estimated according to this model. The EC50 values, standard deviations, and coefficients of determination (r2) were calculated from triplicate experiments. Crystallization. Ten millimolar stock solutions (dimethyl sulfoxide) of Glab were used for the TTR−Glab complex preparation. WT-apo crystals were grown by mixing 2 μL of protein solution (15 mg/mL WT-TTR; 20 mM Tris−Cl, pH 8.0; 150 mM NaCl) with 2 μL of reservoir solution containing 34−40% polyethylene glycol (PEG) 400; 0.1 M HEPES, pH 7.5; and 0.4 M CaCl2 using the hanging-drop vapor diffusion method at 293 K. WT-Glab complex crystals were prepared by soaking WT-apo crystals in a solution containing 1 mM Glab; 34.2% PEG 400; 0.09 M HEPES, pH 7.5; and 0.36 M CaCl2 for 24 h at 293 K. Prior to V30M−Glab crystallization, the V30M solution (9.9 mg/mL V30M-TTR; 20 mM Tris−Cl, pH 8.0; 150 mM NaCl) was incubated for 30 min at room temperature in the presence of 0.5 mM Glab. V30M−Glab crystals suitable for X-ray diffraction experiments were grown from 32% PEG 400; 0.1 M HEPES, pH 7.5; and 0.4 M CaCl2. The typical crystal dimensions were 0.2 mm × 0.05 mm × 0.05 mm. Crystals were directly frozen in liquid nitrogen until data collection. X-ray Diffraction Data Collection, Processing and Structure Refinement. X-ray diffraction data were collected at beamline BL-5A at the Photon Factory or at beamline NW12A at the Photon Factory Advanced Ring in Japan. The diffraction data set of WT-apo was processed with MOSFLM39 and scaled with SCALA,40 and those of WT-Glab and V30M−Glab were processed with HKL2000 and SCALEPACK.41 The X-ray structure of TTR (PDB code: 3U2I)28 was used as the initial model for WT-apo data, and the refined WT-apo structure was used for the Glab complex data. The 3D structure of Glab was obtained from ZINC42 and refined using the PRODRG server.43,44 The dictionary data for Glab were generated using 1094

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PHENIX.ELBOW45 and manually modified if there were any errors. The protein structures were refined using PHENIX.REFINE46 with stepwise cycles of manual model building using COOT.47 The Glab molecule was refined with 50% of the occupancy in each site. The final models were evaluated using the Protein Data Bank validation suite.48 The coordinates and structure factors of WT-apo, WT-Glab, and V30M−Glab have been deposited in the Protein Data Bank under the accession codes 4N85, 4N86, and 4N87, respectively. The omit difference Fourier maps were calculated using CNS SOLVE 1.3.49,50 All structure figures were created using CCP4 mg51 or PyMOL.52

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ASSOCIATED CONTENT

S Supporting Information *

Crystal data and refinement data statistics, semilog plot of the thioflabin T assay, crystal structure around V30/M30. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81 (0)76-434-7595. Fax: +81 (0)76-434-7872. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge access to the synchrotron radiation facility at PF, Japan. This work was supported by the Takeda Science Foundation, Japan, and by a grant for the Amyloidosis Research Committee from the Ministry of Health, Labour, and Welfare, Japan.

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ABBREVIATIONS USED TTR, transthyretin; WT, wild type transthyretin; V30M, V30M mutated transthyretin; FAP, familial amyloid polyneuropathy; FAC, familial amyloid cardiomyopathy; SSA, senile systemic amyloidosis; Glab, glabridin; Equo, (R)-equol; Daid, daidzein; Geni, genistein; Lute, luteorin; Difl, diflunisal; PEG, polyethylene glycol

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REFERENCES

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