Crystal structure determination at 2.3-A resolution of human

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 6644-6648, July 1992

Biochemistry

Crystal structure determination at 2.3-A resolution of human transthyretin-3',5'-dibromo-2',4,4',6-tetrahydroxyaurone complex (thyroid hormone transport proteins/flavonoid/deiodinase/conformation/cocrystal)

EWA CISZAK*, VIVIAN CODYt, AND JOSEPH R. LUFT Molecular Biophysics Department, Medical Foundation of Buffalo, Inc., 73 High Street, Buffalo, NY 14203

Communicated by Herbert A. Hauptman, March 30, 1992

ABSTRACT The crystal structure of the complex of 3',5'dibromo-2',4,4',6-tetrahydroxyaurone, a flavone derivative, with human transthyretin (TTR), a serum thyroid hormone transport protein, has been determined and refined to R = 17.9% for data to 2.3-A resolution and provides a detailed description of a protein-bound flavonoid structure. This bromoaurone is a potent competitor for thyroid hormone binding to TTR, a 54,980-dalton a4 tetrameric protein of 222 molecular symmetry, as well as an inhibitor of iodothyronine deiodinase. Crystals of the TTR-bromoaurone complex are isomorphous to those of native TTR. Interpretation of difference Fourier electron density maps revealed two binding modes for the bromoaurone in each of the two independent binding sites of the TTR tetramer: deep in the channel near Ser-117 (mode I) and near the channel entrance (mode II). None of the binding modes can be fully occupied because of overlap between binding positions. A statistical disorder for bromoaurone binding was also applied, as it binds along the twofold crystallographic axis and does not possess such symmetry. The binding of mode I and that of mode II were refined at half occupancy, resulting in two molecules per tetramer. The bromoaurone binds in a nonplanar antiskewed conformation. The molecular pattern for TTR binding consists of halogen groups able to anchor between fl-sheets to form both hydrophobic and hydrophilic contacts. Comparison of structural data for bromoaurone- and thyroxine-TTR complexes indicates that bromoaurone binding mode I is 3 A deeper in the channel and binding mode II is 4 A further from the channel center than thyroxine. The bromoaurone binding observed in this TTR complex differs significantly from that based upon computer modeling studies.

0 4.

Q ,,

0

-

.'

'.

O_

-o_ \

b

,li 7 4

.

1'

C,

d FIG. 1. Molecular conformations of the thyroid hormones and aurones as typified by planar 3',5'-dibromo-2',4,4',6-tetrahy-

droxyaurone (4' = 00) (a), skewed (4/4' = 900/00) (b), antiskewed 3,3',5'-triiodo-L-thyronine (rT3) (4/4' = 00/90°) (c), and superposition of rT3 and bromoaurone in antiskewed conformation (d).

Human transthyretin (TTR) is one of three plasma proteins responsible for the serum transport of thyroid hormones such as thyroxine (T4), or products of its enzymatic degradation by iodothyronine monoiododeiodinase (ITHD) (1). Recent data have shown that many pharmacological agents are strong competitors with T4 for the hormone-binding sites on TTR and that this competition can interfere with their pharmacological actions (2). In addition, structure-activity studies suggest correlations between the binding affinities of thyroid hormones and competitors for the hormone binding to TTR, as well as their inhibitory potency for ITHD (3). For example, flavonoids, naturally occurring plant pigments that constitute the active ingredients in many folk medicines (4), have been used in the treatment of thyroid disease and shown to be potent cofactor-competitive inhibitors of ITHD (5-7). Furthermore, among tested flavonoids, aurones are the most potent cofactor-competitive inhibitors of ITHD, as well as potent competitors for T4 binding in TTR (8, 9). Activity studies revealed that the ITHD ID50 (1.5 ,uM) for 3',5'-

dibromo-2',4,4',6-tetrahydroxyaurone (Fig. 1) is comparable to the apparent Km value (2 ,uM) of the hormone substrate T4 (5). One hypothesis for the inhibitory potency of aurones in the thyroid deiodinase enzyme system is their ability to mimic the conformational features of thyroid hormones. To understand the thyromimetic action of flavonoids, we compared the crystal structures of the thyroid hormones and selected flavonoids. Structural data show that thyroid hormone conformations fall into two broad classes: skewed with the two phenyl rings perpendicular and bisecting, and antiskewed, as typified by the structures of T4 and rT3 (Fig. 1) (10). Furthermore, the bulk of the ortho tyrosyl iodine atoms prevents T4 from adopting a planar conformation, whereas rT3 may. On the other hand, crystallographic data for aurones show a preference for a planar conformation, as do molecular orbital energy calculations (11, 12). However, the low energy Abbreviations: TTR, transthyretin; T4, thyroxine; rT3, 3,3',5'triiodothyronine; ITHD, iodothyronine monoiododeiodinase. *On leave from Pedagogical University, Institute of Chemistry, ul. Oleska 48, 45-052 Opole, Poland. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6644

Biochemistry: Ciszak et al. barrier to rotation indicates that aurones can adopt a nonplanar conformation. Thus, the best homology between thyroid hormones and flavonoids was obtained by superposition of their respective halogenated phenolic rings (Fig. 1) (9). Structural data for native TTR (13) and the TTR-T4 complex (14) show that the four identical 127-residue monomeric subunits form a tetramer with molecular 222 symmetry. Two hormone-binding sites are located within a channel running through the tetramer along a crystallographic twofold axis that generates the complete binding environment between monomers A and A' and B and B' (Fig. 2). Biochemical data reveal a 100-fold difference in the affinity for binding of the first and second T4 (15). This difference was explained as negative cooperativity, but its mechanism is still unclear. Therefore, the similarity observed between ITHD enzyme inhibition and competitive binding affinity for TTR among thyroid hormones and flavonoids indicated that structural data for the TTR-T4 complex (14) could be used to model the ITHD ligand-binding site. Computer graphic models of their mode of interaction in the T4 binding site of TTR were investigated (5-9). These studies resulted in a model that indicated optimal binding interactions for an antiskewed aurone matched to the T4 (Fig. 1) (9). This model also suggested potentially favorable interactions of the benzofuran ring functional groups with side-chain residues in the hormone-binding site. However, when a planar aurone was modeled, a number of less favorable interactions were present. These data also indicated that more than one orientation of the aurone in the binding channel is possible (9). Therefore, to understand the thyromimetic actions of flavonoids, for both the inhibition of ITHD activity and the competition with T4 binding to TTR, and to understand the mechanism of molecular recognition for different thyroid hormone analogues, we have undertaken a program to determine the crystal structure of several TTR complexes (16, 17). Here we report the results of the structure determination of the TTR-3',5'-dibromoaurone complex.t

MATERIALS AND METHODS Crystallization and Data Collection. TTR was purified from human plasma (18), incubated with 3',5'-dibromo-2',4,4',6tetrahydroxyaurone and phosphate buffer at pH 6.9, and crystallized at room temperature by using the batch technique with 50% saturated ammonium sulfate, 0.1 M sodium phosphate buffer at pH 6.9. Yellow crystals grew in 3-6 weeks as well-shaped prisms. They belong to space group P21212 and have unit cell dimensions of a = 43.40 (1) A, b = 85.84 (1) A, c = 65.78 (1) A, volume = 2.451 (2) x 105 A3. Two monomers per asymmetric unit (monomer volume = 2.23 A3/dalton) correspond to a solvent content of approximately 45% (19). Crystals of the bromoaurone complex are isomorphous to those of native TTR (13) with cell differences of about 1%. A single crystal of dimensions of 0.6 x 0.4 x 0.3 mm was used for data collection with an Enraf-Nonius model CAD4 diffractometer in the 0/20 scan mode with monochromated Cu Ka, radiation (A = 1.54184 A). Data were collected in shells of decreasing in 0/A to minimize effects of crystal decay. The intensities of 4 standard reflections, measured every 250 reflections, showed an average decrease of 5.8%. A q scan was measured and subsequently used to apply an absorption correction (20). Intensities were corrected for Lorentz and polarization effects and for radiation damage and were merged to yield a total of 11,830 independent data with an R-merge of 5.0% for 18,899 measurements (21); 8598 tThe atomic coordinates and structure factors have been deposited in the Protein Data Bank, Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973 (reference 1THC).

Proc. Natl. Acad. Sci. USA 89 (1992)

AAow

6645

-

&~~~~~~~~~~

A

B

FIG. 2. The a-carbon trace of TTR tetramer viewed

across

crystallographic twofold symmetry along axis z. Monomers A and B make up the crystallographic asymmetric unit; A' and B' are twofold symmetry-related along the z axis. The location of the bromoaurone in the two binding sites, AA' and BB', is shown with their van der

Waals surface.

reflections with FO > 2or(FO) and 8442 between 10.0 and 2.3 A (75% of complete) were used. Determination of Bromoaurone Binding. Since these crystals are isomorphous with those of native TTR (13), coordinates of the native protein, reported only for residues 10-123 of the total of 127 residues in each of the two crystallographic independent monomers (2PAB; Brookhaven Protein Data Bank), were used to calculate the phases. Preliminary refinement began with CORELS (22) refining the two independent monomers as rigid groups and resulted in R = 39.1% for 10.0to 3.0-A data. Refinement was continued by using the restrained least-squares program PROLSQ (23, 24) in combination with the model-building program FRODO (ref. 25; modified by J. Sack, J. W. Pflugart, and M. A. Saper, Baylor College of Medicine, Houston). Refinement using an overall B of 16 A2 for data between 10.0 and 2.7 A resulted in R = 25.5%. The refinement was continued, allowing individual isotropic temperature factors to vary. Initial (IF01 - IFcI)exp iac maps, in which FO is the observed structure factor and Fc is the calculated structure factor based on the protein model only, and ac is the calculated phase, produced four peaks above 7oa located in the protein channel along the crystallographic twofold axis, two of them in domain AA' and two in domain BB' (Fig. 3). Each of these peaks had symmetry-related pairs 5.0-5.6 A apart that corresponded to the intramolecular Br-Br distance in bromoaurone. Two modes of bromoaurone binding were considered: one with the bromophenolic ring positioned near the channel center and the benzofuran fragment toward the channel entrance, defined as forward binding mode (I), and another with the bromophenolic ring near the channel entrance and the rest of molecule accommodated within the channel interior, described as reverse binding. However, in the reverse binding mode the bromoaurone made short contacts (=1.0 A) to main-chain atoms of a-strands and when included in the refinement resulted in peaks with negative density in its (21FOI - lFcl)exp iac electron density map. Instead, a second forward binding mode was considered in which the bromoaurone is located close to the channel entrance with the bromophenolic fragment in the channel and the benzofuran fragment close to the protein surface (II) (Fig. 3).

Biochemistry: Ciszak et al.

6646


a

b

FIG. 3. Stereo view across the crystallographic twofold axis of the TTR tetramer indicating the highest electron density peaks, contoured at 7a, from the initial (1F01 - IFcI)exp id, map calculated on only the protein model. For clarity, a shows the fit of the bromoaurone to the peaks near the channel center, binding mode I, and b illustrates the fit to the peaks closer to the channel entrance, binding mode H. The twofold related position of the bromoaurone is shown in thinner lines.

None of the bromoaurone positions can be fully occupied because of overlap between the binding of modes I and II. In addition, because bromoaurone binding is along the channel axis, which is coincident with the crystallographic twofold symmetry axis, a statistical disorder model with half occupancy was applied. This results in two bromoaurone molecules per tetramer. Refinement. Further restrained refinement was continued, including the bromoaurone molecule. The PROLSQ program was modified to ignore the close contacts resulting from the twofold symmetry. Between least-squares minimizations the structure was manually adjusted to its (21Fol - IFcI)exp iac maps, and these adjustments were verified by calculation of series of maps with deleted fragments of protein or competitor molecule. The highest thermal vibrations of the protein residues are in regions near the channel entrance. However, no interpretable electron density was observed for the N-terminal residues 1-8 in monomer A and 1-9 in monomer B or for the C-terminal residue 127 in monomer A and 125-127 in monomer B. Since the benzofuran fragment of bromoaurone bound in mode H is located in that region it was not included in the final refinement (Fig. 3). Values of the bromoaurone thermal parameters for atoms included in the refinement are about 10% higher than the average isotropic thermal parameters for TTR. In the final refinement stages water molecules were added in accord with the criteria of good electron density and acceptable H bonds to other atoms. The refinement was completed at an R factor of 0.179, for 8442 data points [Fo > 2of(Fo)] between 10.0- and 2.3-A resolution with a mean temperature factor for main-chain atoms of 19.2 A2 and all-chain atoms of 21.2 2. The side chains of Glu-62 in monomers A and B were refined in two

alternative conformations. A summary of the target and observed deviations from ideal geometry is given in Table 1. The final model of TTR-bromoaurone complex consists of coordinates and temperature factors for residues 9-126 in monomer A, residues 10-124 in monomer B, two bromoaurone molecules per tetramer, and 95 well-defined water molecules.

Table 1. Summary of geometrical parameters for the TTR-bromoaurone complex rms ao deviation of Target a, value Parameter model from target Distances, A Bond distances 0.020 0.017 0.040 0.051 Angle distances Planar 1-4 distances 0.050 0.063 Planar groups, A 0.020 0.016 Chiral volume, A3 0.150 0.197 Nonbonded distances, A 0.500 Single torsion 0.236 0.500 Multiple torsion 0.292 Possible hydrogen bonds 0.500 0.265 Torsion angles, Planar 5.0 3.3 15.0 25.1 Staggered Orthonormal 18.1 15.0

7 x 10 A, which is occupied by four water molecules (Fig. 3). Since the topologies of the binding sites AA' and BB' and their bromoaurone interactions are similar, the detailed description ofbromoaurone binding interactions will be given in terms of one binding site (Fig. 5). Table 2 lists the contacts in both binding sites for the two binding modes. In the case of deep forward binding (I) the bromoaurone is involved in a series of hydrophobic and hydrophilic interactions with surrounding residues Thr-119, Ser-117, Leu-110, Ala-109, Ala-108, Leu-17, and Lys-15. As illustrated in Fig. 5, the bromophenolic fragment is between residues 108-110 and 117-119. The bromine atoms are accommodated within symmetry-equivalent P-strand pockets composed of mainand side-chain atoms of Ala-108, Ala-109, Ser-117, Thr-118, and Thr-119, which provide hydrophilic and hydrophobic contacts. The shortest hydrophilic bromine contacts are formed to the OY of Ser-117 (3.1 A in AA' and 3.2 A in BB'). These distances are in the range of Br *0 contacts found in other structures (26, 27). The shortest bromine hydrophobic

RESULTS Overal Stuture. The chemically identical but structurally independent monomers A and B make up the asymmetric unit, and the location of the crystallographic twofold axis along z produces the tetramer. Within each monomer the peptide chain adopts similar conformations. As illustrated in Fig. 4, the largest differences in conformation between the two monomers exist in the loop fragments 36-39, 57-63, and 97-103, which are located on the protein surface near the channel entrance and between the N- and C-terminal residues. These fragments are characterized by high thermal motions among the whole protein. The rms deviation between main-chain atom positions of all residues in both monomers is 0.7 A. When an analogous alignment was limited to main-chain atoms in only the P-strand residues, the rms difference was 0.4 A. The rms deviation in the alignment of main-chain atoms of complexed and native protein dimers is 0.5 A, indicating the conserved nature of the protein dimers themselves. Thus, the tertiary TTR structures in complexed and native forms are nearly identical. Binding Features. The binding channel that runs through the whole protein tetramer is composed of only p-strand residues and has a fairly constant diameter of about 10 A. Two narrow constrictions of 5 A made by the side chains of Ser-117 of monomers A and B and their symmetry-related pairs create a central cavity at the tetramer interface of 7 x



6647

FIG. 4. Stereo view of the superposition of the main-chain atoms of monomer A (solid lines) on monomer B (broken lines). The N and C termini, regions of significant differences, and residues that make direct contacts to the bromoaurone ligand are identified.

interactions are to CY2 of Thr-119 (3.1 and 3.2 A). The bromoaurone 4'-OH group forms hydrogen bonds to Oy of Ser-117 or its symmetry equivalent of 3.3 A in AA' and 3.4 A in BB'. The 2'-OH group also makes favorable contacts to O'1 of Thr-119. The benzofuran system of the aurone is in a hydrophobic patch of the binding site between Ala-108, Ala-109, Leu-17, and Lys-15. Its 4-OH or 6-OH groups can hydrogen bond to the protonated amino group of Lys-15 or to the 0"1 of Thr-106. The best fit of the bromoaurone to the observed electron density indicates that it has a nonplanar conformation with a torsion angle of about 45°, similar to the energy-minimized structure (12). Docking of the bromoaurone at the channel entrance in mode II is not as tight as for mode I, and its binding is characterized by fewer close contacts (Fig. 6). The bromophenolic fragment binds in a hydrophobic patch of the channel, with the bromine atoms occupying symmetryequivalent pockets lined with the methyl groups of Thr-106 and Ala-108 and methylene groups of Lys-15 and Leu-17. The bromine atoms also make Br ..C contacts of 3.5-3.9 A (Table 2). Since no interpretable electron density was observed in the region near the channel entrance where the benzofuran fragment is projected, no contribution for it was included in the refined complex.

DISCUSSION To understand binding of thyromimetic compounds to TTR and to delineate their common features, bromoaurone bind-

FIG. 5. Bromoaurone in binding mode I (thick lines) superimon a final (IFOI IFCI)exp iac electron density calculated without bromoaurone molecules contoured at 3oa. The symmetryrelated bromoaurone is shown in lighter lines. For clarity, the contribution of the bromophenolic ring of binding mode H to the density is not shown. Protein residues are twofold related.

posed

-

ing was compared with that of T4. As shown in Fig. 6, T4 binds in a skewed conformation with its iodophenolic ring bound deep in the channel and oriented about 450 with respect to the (3-sheets of both monomers (14). In this orientation the 3',5'-phenolic iodine atoms do not occupy symmetryequivalent pockets on both sides of channel /3-sheets. The 3'-I is located in the pocket formed by Ala-108, Ala-109, Leu-110, Lys-17, and Lys-15, forming tight hydrophobic and hydrophilic interactions with the surrounding atoms. The 5'-I atom is on the opposite side of the channel in a different environment near a pocket formed by Ala-108, Ala-109, Leu-110, Ser-117, and Thr-119. When bromoaurone binds in mode I, the 3',5'-Br atoms are located in symmetryequivalent pockets surrounded by Ala-108, Ala-109, Leu110, Ser-117, Thr-118, and Thr-119. Thus the bromine atoms bind deeper in the channel than the T4 iodine atoms. A similar binding orientation was observed in the 3,3'-diiodothyronine-TTR complex (16). In the T4 complex these symmetryequivalent pockets are occupied by water, which mediates the interactions between the phenolic ring and Ser-117. In the case of binding mode II, the bromine atoms occupy the same hydrophobic environment as the 3,5-tyrosyl iodine atoms of T4 but are -1.5 A closer to the channel center. Table 2. Close contacts characterizing bromoaurone-protein interactions for binding modes I and II Distance, A Ligand Ligand atomt Monomer A Monomer B Residue Atom atom* Binding mode I 2.7 NC 3.1 041 Lys-15 3.5 3.2 041 NC Lys-15 Co 2.6 2.6 02' Ala-108 Co 4.1 3.9 C6' Ala-108 0Y 3.1 3.2 Ser-117 Brl 0Y 3.2 3.4 Br2 Ser-117 4.0 3.7 Brl Ser-117 0 4.0 Br2 3.9 0 Ser-117 0Y 3.4 3.3 04' Ser-117 3.4 04' 3.3 Ser-117 0r 3.5 3.9 Thr-118 C Brl 3.9 4.1 Br2 Thr-118 C 3.7 3.9 N Thr-118 Brl 3.8 Br2 4.3 N Thr-118 3.2 3.1 Thr-119 Cy2 Brl 3.4 3.2 Br2 Thr-119 C"2 3.4 2.6 02' Thr-119 O'Y Binding mode H 3.0 2.9 02' NC Lys-15 C'61 3.6 3.5 Leu-17 Brl C81 3.5 3.5 Br2 Leu-17 CP 3.6 3.9 Ala-108 Brl *Symmetry code: x, y, z. tSymmetry code: -x, -y, z.

11118



FIG. 6. Stereo view comparison of the binding orientation of T4 (broken lines) with that of bromoaurone binding in mode I (thick lines) and mode II (thin lines) in the binding site viewed across the long axis of the channel. Halogen atoms of T4 and bromoaurone are emphasized as squares for I and circles for Br. For clarity, the alternative twofold symmetry-related positions of T4 and bromoaurone have not been shown.

Bromoaurone in binding mode I forms more extensive interactions inside the TTR cavity than T4. However T4, which is a longer molecule and does not bind as deep as bromoaurone, has ion-pair interactions at the channel entrance between its carboxyl and amino groups and the terminal groups of residues Lys-15 and Glu-54. In the bromoaurone complex, Glu-54 is too far to be involved in direct hydrogen bonds and Lys-15 can interact only with the 6-OH group of bromoaurone. An interesting feature of both bromoaurone and T4 complexes is that the phenolic halogens are tightly anchored within the a-strand pockets and form short (I3' O0 of Ala-109 3.0 A; Br ..* .OQ of Ser-117 = 3.2 A in AA' and 3.1 A in BB') interactions (Table 2). Such close contacts between halogens and oxygen have been observed in other x-ray crystallographic studies including the thyroid hormones (27), and they may indicate either strong electrostatic interactions or charge-transfer interactions. One of the most important consequences resulting from the formation of a charge complex could be the change in the electronic characteristics of interacting residues that may play an important role in the cooperative action of monomers. The molecular pattern for TTR binding consists of halogen groups able to anchor in the deep binding grooves between P3-sheets to form both hydrophobic and hydrophilic contacts. The recognition and exploitation of 8-strand pockets of the channel permit tight binding that presumably enhances binding affinity to TTR. These structural data further reveal that in this TTR-bromoaurone complex the orientation of the bromoaurone differs significantly from that proposed from computer graphic modeling studies (9). The bromoaurone binds not only about 3 A deeper in the channel and with a different orientation than T4 but also in more than one mode. In contrast to the planar structures observed for aurones (11, 12), the conformation of bromoaurone in binding mode I is nonplanar. Furthermore, these studies reveal the presence ofalternative halogen-binding sites within the hormone channel, which provide insight into understanding the mechanism of molecular recognition for thyroid hormone and competitor binding to TTR. Thus, knowledge of these structural details can be useful in the design of novel compounds with thyromimetic action. Explanation of biochemical data showing that bromoaurone is a potent type I ITHD inhibitor, as well as a strong competitor for T4 binding to TTR, lies in the ability of the aurones to mimic thyroid hormone conformational features. Thus, TTR binding interactions could provide a useful model of ITHD, which binds rT3 with a higher affinity than T4 (8, 9). This study reveals that, indeed, there is a greater homology in the conformation of bound bromoaurone for rT3 than for T4 (Fig. 1) and further suggests that the presence of the tyrosyl ring halogens is less critical to deiodinase binding than to TTR. This conclusion is supported by the observation of a second bromoaurone molecule bound with its halogens occupying the tyrosyl 3,5-I positions of T4.

We thank Dr. Paul J. Davis (Medical College of Albany) for assistance with TTR purification procedures, Dr. Josef Koehrle (Hannover, F.R.G.) for his interest, Dr. K. Irmscher (Merck, Darmstadt, F.R.G.) for samples of the bromoaurone, Dr. Walter Pangborn (Medical Foundation of Buffalo) for data collection, and Dr. G. David Smith (Medical Foundation of Buffalo) for his helpful discussions during refinement and use of his programs EMPABS for empirical absorption corrections and FITMOL for least-squares fit of molecules. This research was supported in part by National Institutes of Health Grant NDDK41009. 1. Robbins, J. (1991) in The Thyroid, eds. Braverman, L. E. & Utiger, R. D. (Lippincott, New York), pp. 111-125. 2. Cavaliere, R. R. & Pitt-Rivers, R. (1981) Pharmacol. Rev. 33, 55-80. 3. Koehrle, J., Hesch, R. D. & Leonard, J. L. (1991) in The Thyroid, eds. Braverman, L. E. & Utiger, R. D. (Lippincott, New York), pp. 144-189. 4. Cody, V., Middleton, E., Jr., Harborne, J. B. & Bertz, A. (1988) Plant Flavonoids in Biology and Medicine II: Biochemical, Cellular and Medicinal Properties (Liss, New York). 5. Koerhle, J., Auf'mkolk, M., Spanka, M., Sonyi, G., Hesch, R.-D. & Cody, V. (1985) in Flavonoids and Bioflavonoids, eds. Farkas, L., Gabor, M. & Kallay, F. (Hungar. Acad. Sci., Szeged), pp. 411-421. 6. Koehrle, J., Auf'mkolk, M., Spanka, M., Irmscher, K., Cody, V. & Hesch, R.-D. (1986) in Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and Structure-Activity Relationships, eds. Cody, V., Middleton, E., Jr., & Harborne, J. B. (Liss, New York), pp. 359-371. 7. Koehrle, J., Spanka, M., Irmscher, K. & Hesch, R.-D. (1988) in Plant Flavonoids in Biology and Medicine 11: Biochemical, Cellular and Medicinal Properties, eds. Cody, V., Middleton, E., Jr., Harborne, J. B. & Beretz, A. (Liss, New York), pp. 323-340. 8. Koehrle, J., Auf mkolk, M., Rokos, H., Hesch, R.-D. & Cody, V. (1986) J. Biol. Chem. 261,11613-11622. 9. Auf mkolk, M., Koehrle, J., Hesch, R. D. & Cody, V. (1986) J. Biol. Chem. 261, 11623-11630. 10. Cody, V. (1980) Endocrine Rev. 1, 141-160. 11. Cody, V. (1988) in Plant Flavonoids in Biology and Medicine 11: Biochemical, Cellular and Medicinal Properties, eds. Cody, V., Middleton, E., Jr., Harborne, J. B. & Beretz, A. (Liss, New York), pp. 29-44. 12. Ciszak, E., Cody, V., Luft, J., Kempton, R. J. & Kesler, B. S. (1991) J. Mol. Struct. (Theochem) 231, 345-357. 13. Blake, C. C. F., Geisow, M. J., Oatley, S. J., R6rat, B. & Rerat, C. (1978) J. Mol. Biol. 121, 339-356. 14. Blake, C. C. F. & Oatley, S. J. (1977) Nature (London) 268, 115-120. 15. Pages, R. A., Robbins, J. & Edelhoch, H. (1973) Biochemistry 12,

2773-2779.

16. Wojtczak, A., Cody, V. & Luft, J. (1992) J. Biol. Chem. 267, 353-357. 17. Cody, V., Wojtczak, A., Ciszak, E. & Luft, J. (1991) in Progress in Thyroid Research, eds. Gordon, A., Gross, J. & Hennemann, G. (Balkema, Rotterdam, The Netherlands), pp. 793-796. 18. Tritsch, G. L. (1972) J. Med. 3, 129-145. 19. Matthews, B. W. (1968) J. Mol. Biol. 33, 491-497. 20. North, A. C. T., Phillips, D. C. & Mathews, F. S. (1968) Acta Crystallogr. Sect. A 24, 351-359. 21. Blessing, R. H. (1989) J. Appl. Crystallogr. 22, 396-397. 22. Sussman, J. L., Holbrook, S. R., Church, G. M. & Kim, S.-H. (1977) Acta Crystallogr. Sect. A 33, 800-804. 23. Hendrickson, W. A. & Konnert, J. H. (1980) in Computing in Crystallography, eds. Diamond, R., Ramaseshan, S. & Venkatesan, K. (Indian Acad. Sci., Bangalore), pp. 13.01-13.23. 24. Finzel, B. C. (1987) J. Appl. Crystallogr. 20, 53-55. 25. Bush, B. L. & Jones, T. A. (1988) FRODo, Molecular Graphics Program for the PS300 (Uppsala Univ., Uppsala). 26. Murray-Rust, P. & Motherwell, W. D. S. (1979) J. Am. Chem. Soc. 101, 4374-4376. 27. Cody, V. & Murray-Rust, P. (1984) J. Mol. Struct. 112, 189-199.