Interspecies Variation Between Fish and Human Transthyretins in

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Ecotoxicology and Human Environmental Health

Interspecies Variation Between Fish and Human Transthyretins in Their Binding of Thyroid-Disrupting Chemicals Jin Zhang, Christin Grundström, Kristoffer Brannstrom, Irina Iakovleva, Mikael Lindberg, Anders Olofsson, Patrik L. Andersson, and A. Elisabeth Sauer-Eriksson Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03581 • Publication Date (Web): 18 Sep 2018 Downloaded from http://pubs.acs.org on September 18, 2018

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Environmental Science & Technology

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Interspecies Variation Between Fish and Human

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Transthyretins in Their Binding of Thyroid-

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Disrupting Chemicals

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Jin Zhang†, Christin Grundström†, Kristoffer Brännström‡, Irina Iakovleva†, Mikael Lindberg†,

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Anders Olofsson‡, Patrik L. Andersson†*, A. Elisabeth Sauer-Eriksson†*

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†Department of Chemistry, Umeå University, SE-90187 Umeå, Sweden

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‡Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå,

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Sweden

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Corresponding Authors

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*Email: [email protected]. Phone: +46-(0)907865266

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*Email: [email protected]. Phone: +46-(0)907865923

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ACS Paragon Plus Environment

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ABSTRACT

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Thyroid-disrupting chemicals (TDCs) are xenobiotics that can interfere with the endocrine

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system and cause adverse effects in organisms and their offspring. TDCs affect both the thyroid

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gland and regulatory enzymes associated with thyroid hormone homeostasis. Transthyretin

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(TTR) is found in serum and cerebrospinal fluid of vertebrates, where it transports thyroid

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hormones. Here we explored the interspecies variation in TDC binding to human and fish TTR

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(exemplified by Gilthead seabream (Sparus aurata)). The in vitro binding experiments showed

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that TDCs bind with equal or weaker affinity to seabream TTR than to the human TTR,

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particular, the polar TDCs (>500-fold lower affinity). Crystal structures of seabream TTR–TDC

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complexes revealed that all TDCs bound at the thyroid binding sites. However, amino acid

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substitution of Ser117 in human TTR to Thr117 in seabream prevented polar TDCs from binding

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deep in the hormone binding cavity, which explains their low affinity to seabream TTR.

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Molecular dynamics and in silico alanine scanning simulation also suggested that the protein

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backbone of seabream TTR is more rigid than the human one and that Thr117 provides fewer

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electrostatic contributions than Ser117 to ligand bindings. This provides an explanation for the

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weaker affinities of the ligands that rely on electrostatic interactions with Thr117. The lower

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affinities of TDCs to fish TTR, in particular the polar ones, could potentially lead to milder

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thyroid-related effects in fish.

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INTRODUCTION

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Thyroid-disrupting chemicals (TDCs) are environmental xenobiotics that may induce significant

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disorders in physiological processes in humans and wildlife, including macronutrient

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metabolism, energy balance, brain development, and reproduction.1-3 They can alter the structure

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or function of the thyroid gland, interfere with thyroid hormone signaling, and subsequently

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disrupt its homeostasis.4 TDCs have multiple molecular mechanisms. For example, hydroxylated

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polychlorinated biphenyls (OH-PCBs) bind to and disturb thyroid hormone receptors and

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transthyretin,5-7 and polyhalogenated aromatic hydrocarbons accelerate the clearance of thyroid

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hormones by inducing uridine diphosphoglucuronosyl transferases.8 Even low exposures to

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TDCs are cause of concern as these can trigger non-monotonic dose-response relationships

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resulting in severe adverse effects.9-12 In coastal regions with high fish consumption TDC levels

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have been associated with increased incidence of thyroid cancer and thyroid autoimmune

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disease.13, 14

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Transthyretin (TTR) is a serum transport protein that delivers 3,3',5-triiodo-L-thyronine (T3)

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and 3,3',5,5'-tetraiodo-L-thyronine (thyroxine T4) from the thyroid gland to target tissues in

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vertebrates.15 TTR is a molecular transporter of TDCs that include per- and polyfluoroalkyl

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substances (PFASs)16 and OH-PCBs.17 TDCs in the thyroxine-binding sites (TBSs) of TTR can

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disrupt TTR-mediated thyroid hormone transport.18 The subsequent accumulation of TDCs in

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vital organs19 by TTR-assisted delivery, may cause developmental disorders.20

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The three-dimensional structure of transthyretin shows it to be a homotetramer with a central

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hydrophobic channel in which two TBSs are situated.21, 22 Over 200 human TTR (hTTR) crystal

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structures have been deposited in the Protein Data Bank (PDB).23,

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Many of these are


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complexes, of which some are complexed with environmental pollutants or their metabolites,

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including

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tetrahydroxybenzophenone (BP2), perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid

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(PFOS), tetrabromobisphenol A (TBBPA), and hydroxylated polychlorinated biphenyls (OH-

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PCBs)18, 25, 26, which are of relevance for this study.

hTTR

complexes

with

4,4'-sulfonyldiphenol

(bisphenol

S),

2,2’,4,4’-

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TTR transports TH via the choroid plexus into the brain in fish27 and it is expressed in the

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liver, brain, eye, and heart of fish larvae from the first day post-hatch.28 Data on TTR-TDC

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complexes from fish species are scarce despite their importance in toxicity studies.29, 30 Gilthead

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seabream (Sparus aurata) is an indicator species for the estimation of environmental pollutant

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levels in aquatic environments.31 It is one of the most consumed fish species and it is widely

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distributed in the Mediterranean sea and the eastern Atlantic ocean.32 Several classical TDCs

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have been detected in seabream in concentrations ranging from 36.2 pg/g to 141 ng/g wet

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weight, e.g. polychlorinated dibenzo-p-dioxins, dibenzofurans, PCBs, and polybrominated

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diphenyl ethers (PBDEs).33 TDCs can disturb thyroid hormone homeostasis in seabream via

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binding to TTR,34 the primary thyroid hormone transporter; this may cause underdevelopment of

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the juveniles.35 More importantly, TDCs can accumulate in aquatic ecosystems and thereby

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expose humans to high levels of TDCs.36

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TTRs from fish have low sequence identity with hTTR.28, 37 Seabream TTR (SaTTR) has 54%

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sequence identity with hTTR,28 but the only amino acid difference within the TBS is the

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conservative substitution of Ser117 in hTTR to Thr117 in SaTTR.38 Crystal structures of wild-

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type SaTTR38, 39, and SaTTR in complex with T3 and T4 are available.38 However, additional

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crystal structures of the SaTTR protein in complex with diverse environmental pollutants would

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improve our understanding of TDC interaction with SaTTR; interspecies variations of the TTR-


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TDC molecular interactions; and a comparison of the chemical-induced thyroid disruptions in

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fish vs. that in humans.

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Six representative TDCs were selected for this study based on their use in consumer goods and

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products, their structural diversity, and the environmental relevance.16, 25, 40, 41 The compounds

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included TBBPA, BP2, PFOA, 3,5,6-trichloro-2-pyridinol (TC2P), 2,4,5-trichlorophenoxyacetic

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acid (2,4,5-T), and 2-(3-chloro-2-methylanilino)pyridine-3-carboxylic acid (clonixin).16,

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TBBPA is widely used as a flame retardant in electronics. It has been detected in the serum of IT

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technicians and electronics recycling workers (500-fold, Table 1)

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The TTR-TC2P complex structures are of particular interest. Two TC2P molecules bind

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simultaneously within the TBSs in the hTTR-TC2P complex, i.e., one molecule forms a

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hydrogen bond with Ser117 at the cavity bottom, and the second molecule forms a hydrogen

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bond with the Lys15 at the cavity entrance (Figure 3e). This binding mode is similar to that of

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2,6-dinitro-p-cresol (DNPC) in the hTTR,25 which is probably associated with their relatively

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small molecular sizes and their polar nature. In SaTTR, TC2P only binds in the vicinity of the

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side chain of Lys15 at the cavity entrance. The interspecies differences in binding modes are

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consistent with the 10-fold weaker binding affinity of TC2P to SaTTR (Table 1).

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Molecular dynamics simulations for TTR-TDCs interaction studies. We investigated the

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TTR-TDCs molecular interactions by performing molecular dynamics simulations on the human

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and seabream structures in complex with each of the six TDCs.25, 26 All co-ligands remained

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bound in the thyroid-binding sites, and the root-mean-squared-deviations of the protein and

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ligands were under 2 and 3 Å (except for TC2P in SaTTR) throughout the simulations (Figure

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S2). The ligand binding free energies were calculated using the MM-PBSA method based on the

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last 10 ns of the molecular dynamics trajectory.

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We compared the calculated ligand binding free energy both intra- and interspecies. The

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calculated binding free energies and the experimental binding free energy (∆G) are moderately


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correlated yielding R2=0.56 for SaTTR and R2=0.62 for hTTR (Figure S3). This correlation

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suggests that MM-PBSA could be used for estimating the ligand binding free energy of TDCs to

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TTR and as a scoring function to identify novel TDCs and study their molecular interactions.67

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The mean value (-24.4 kcal/mol) of calculated binding free energies of the six TDCs to SaTTR is

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lower than those (-26.2 kcal/mol) for the hTTR (Figure S3), which agrees with the weaker in

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vitro affinities of the ligands (except PFOA) to SaTTR compared to hTTR.

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We tried to elucidate the contributions of residue 117 to the binding free energies by

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performing in silico alanine scanning simulations on the residue in the TTR-TDC complexes.

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TTR-PFOA and TTR-BP2 complexes from the two species were chosen as representatives for

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the simulations, as they have the smallest and largest interspecies variations in in vitro ligand

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binding affinities, respectively.

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The in silico alanine scanning on TTR-PFOA complexes (Figure S4) showed that the

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calculated binding free energy of PFOA to hTTR and SaTTR both became slightly stronger, at a

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similar scale, when residue 117 was mutated to alanine. The slight increase might be due to the

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improvement of the existing hydrophobic interactions between the fluorinated tail of PFOA and

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the hydrophobic cavity of TTR.25 The similarity in scale increase may indicate that Thr117 in

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SaTTR and Ser117 in hTTR provide a similar contribution to the binding of PFOA; this could

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explain the interspecies similarities in in vitro ligand binding free energies. In silico alanine

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scanning simulations of TTR-BP2 complexes resulted in clear interspecies variations in

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mutation-caused shifts of the calculated ligand binding free energies (Figure S4). In the hTTR-

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BP2 complex, we observed a significant decrease (4 kcal/mol) in calculated binding free energy

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of the ligand to mutated hTTR, since mutation of Ser117 causes loss of important electrostatic

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interactions for hTTR-BP2 recognition.25 In the SaTTR-BP2 complex, the mutation posed a


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limited impact on the calculated binding free energy of BP2 to SaTTR, as Thr117 provides fewer

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electrostatic contributions to the ligand binding due to the locked rotamer of its side chain.

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Interspecies variations in mutation-caused shifts of calculated binding free energy could explain

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the significantly weaker in vitro binding free energies of BP2 to SaTTR compared to hTTR

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(Table 1). This supports our conclusions from the crystal structure analysis that Thr117 provides

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fewer electrostatic contributions and that SaTTR has a clear preference to hydrophobic ligands.

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DISCUSSION

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In the present study, we aimed to elucidate the interspecies variations in human and seabream

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TTR-TDC interactions using crystallographic, in vitro, and in silico data. The six TDCs studied

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here have been detected in humans, wildlife, and the environment.

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In vitro isothermal titration calorimetry binding experiments showed that all six TDCs bound

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to SaTTR and hTTR. TBBPA was the strongest TTR binder, with nM affinity to hTTR.26, 68 With

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the exception of PFOA, the TDC binding affinities to hTTR were significantly stronger than to

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SaTTR. The sequence of hTTR and SaTTR differs at the thyroid-binding site, where it is Ser117

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for hTTR and Thr117 for SaTTR. Serine and threonine are both slightly polar amino acids with a

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hydroxyl group at the Cβ position, with the only difference between being that threonine has a

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methyl group in place of the hydrogen group found in serine. A serine-to-threonine substitution

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could therefore be considered as a fairly neutral substitution. However, the structural comparison

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of the thyroid-binding sites in hTTR and SaTTR showed that the substitution has large functional

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consequences for the two proteins. The side chain of residue 117 is situated at both the

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monomer-monomer and the dimer-dimer interfaces. Superimposing all TTR-TDC complexes

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shows that Ser117 in hTTR has high rotational freedom and that its site chain can occupy all


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three possible rotamers (g+, t, and g). In contrast, the Thr117 in SaTTR is rigid and can only

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occupy one rotamer, otherwise steric clashes will occur between the Cγ2 atoms of the rotamers

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(Figure S5). Consequently, the mobility of Ser117 allows hTTR to make good binding

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interactions with both polar and hydrophobic ligands, whereas SaTTR has a clear preference for

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hydrophobic ones. As a consequence, larger ligands, such as T4, BP2 and clonixin, cannot reach

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as deep into the thyroid-binding cavity of SaTTR as they can in hTTR, due to steric clashes with

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the Cγ2 atom of Thr117.

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In addition to this constraint, the rotamer of Lys15, positioned at entrance of the hormone-

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binding channel, is also locked in SaTTR (Figure S5). Its ε-ammonium group forms a hydrogen

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bond with the main chain carbonyl oxygen of Thr52 and a salt bridge with the side chain of

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Glu54. These contacts are present in all monomers of all SaTTR structures solved to date. In

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hTTR, however, the side chain of Lys15 has more rotational freedom (Figure S5). Inspection of

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the hydrophobic core positioned directly underneath Lys15 suggests that the amino acid

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substitutions Val16Ile and Ile73Phe in SaTTR shift the position of the β-strand A (residues

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Leu12-Ala19) by up to 1 Å, which explains the different properties of the hTTR and SaTTR

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Lys15 side chains. The significantly wider entrance of the hormone-binding channel in SaTTR,

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in combination with its narrower cavity, provides an additional structural explanation for the

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different binding affinities of human and seabream TTR to TDCs.38

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The finding that Thr117 in SaTTR prevents high affinity binding to polar ligands could be

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generalized to others piscine species, e.g. zebrafish (Danio rerio), Atlantic salmon (Salmo salar),

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Rainbow trout (Oncorhynchus mykiss), and Channel catfish (Ictalurus punctatus). Like

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seabream, they share high similarity in their thyroid-binding site residues and the same Thr117


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residues.69 Here in silico molecular dynamics simulations could be used with advantage to

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predict potential TDC binders to other fish species TTRs.

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Of the six TDCs used here, BP2 was the most polar. We therefore used isothermal titration

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calorimetry to determine binding affinities of three additional polar compounds to SaTTR. These

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were two thyroid disrupting compounds 2,6-dinitro-p-cresol (DNPC) and bisphenol S (BPS), and

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the flavonoid luteolin (Figure S6a). The binding affinities (Kd) and binding interactions to hTTR

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are known: DNPC 1.3 µM, PDB code 5L4F; BPS 52 µM, PDB code 5L4J; and luteolin 0.07 µM

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PDB code 4QXV.25, 70 These have 34-fold, 1.5-fold, and 250-fold higher affinities, respectively,

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to hTTR than SaTTR. A close look at the hTTR-DNPC, hTTR-PBS, and hTTR-luteolin complex

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structures explains the large variation in binding affinities of the three polar compounds to

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SaTTR, in particular to the 250-fold higher affinity of luteolin to hTTR (Figure S6b). In

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summary, isothermal titration calorimetry data from the three polar compounds support our

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observation that Thr117 prevents high-affinity binding of polar ligands to SaTTR, and

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presumably to other fish species that have a threonine at position 117. The ability of small,

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strong binders adapting to multiple conformations in the thyroid-binding sites can explain the

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interactions between TTR and such ligands, and provide guidance for molecular design and

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hazard identification. Our results also highlight the difficulty in extrapolating data from one

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species to another. Without structural data it would be difficult, if not impossible, to explain the

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differences in the binding affinity data.

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ENVIRONMENTAL IMPLICATIONS

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The thyroid hormone system is vital for early development, metabolism, and growth in wildlife

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and humans. In humans, thyroxine can be transported in the blood by albumin, thyroxine binding


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globulin and TTR, whereas in fish TTR is the primary thyroid hormone transporter.71 It is well-

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known that a large number of environmental pollutants bind to TTR and a few of these are even

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stronger binders than the natural hormone thyroxine.41, 68, 72-74 This specific binding of organic

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contaminants to TTR pose a risk of transport across biological barriers, e.g. the blood brain

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barrier and placenta, and tissue specific accumulation, that could induce adverse effects in

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vulnerable organs or developing organisms. Currently, no specific biomarker for thyroid

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hormone disruption has been generally agreed upon even if effects on hormonal levels, gene

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expression and histology are often being used.1 Binding to TTR is one potential marker of TDCs

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which could be designed for high throughput screening.75 This study described on a molecular

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level potential variation in interaction at the TTR binding site that that could cause species

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variation in read-out from toxicity assays. In particular, these variations are expected for small

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and polar chemicals. In assessing environmental and human health risks of industrial chemicals

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and development of drug candidates, fish based assays is frequently being used motivated by the

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high number of orthologs for human drug targets in e.g. zebrafish.76 Here we studied, seabream

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but the amino acid sequence in the binding pocket of TTR is well conserved for several fish

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species including zebrafish and rainbow trout which are frequently being used for toxicity tests.

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Findings presented here may thus have an impact on data from fish tests for in particular small

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and polar chemicals that could result in underestimating risks when extrapolated to the human

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situation.

376 377

SUPPORTING INFORMATION

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This information is available free of charge via the Internet at http://pubs.acs.org.


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Details of the X-ray structures, in vitro isothermal titration calorimetry experiments; procedures

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and results of the molecular dynamics simulations, MM-PBSA calculations and alanine scanning

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simulations are in the supporting information.

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ACKNOWLEDGEMENT

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This study was financed by the MiSSE project through grants from the Swedish Research

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Council for the Environment, Agricultural Sciences and Spatial Planning (Formas) (210-2012-

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131) and by the Swedish Research Council (521-2011-6427 and 2015-03607). We also thank the

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staff of the Protein Expertise Platform at Umeå University for cloning services. The molecular

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dynamics simulations were conducted at the High Performance Computing Center North.

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49. Fortenberry, G. Z.; Meeker, J. D.; Sanchez, B. N.; Barr, D. B.; Panuwet, P.; Bellinger, D.; Schnaas, L.; Solano-Gonzalez, M.; Ettinger, A. S.; Hernandez-Avila, M.; Hu, H.; Tellez-Rojo, M. M., Urinary 3,5,6-trichloro-2-pyridinol (TCPY) in pregnant women from Mexico City: distribution, temporal variability, and relationship with child attention and hyperactivity. International journal of hygiene and environmental health 2014, 217, (2-3), 405-12. 50. Fortenberry, G. Z.; Hu, H.; Turyk, M.; Barr, D. B.; Meeker, J. D., Association between urinary 3, 5, 6-trichloro-2-pyridinol, a metabolite of chlorpyrifos and chlorpyrifos-methyl, and serum T4 and TSH in NHANES 1999-2002. The Science of the total environment 2012, 424, 351-5. 51. Van den Berg, K. J.; van Raaij, J. A.; Bragt, P. C.; Notten, W. R., Interactions of halogenated industrial chemicals with transthyretin and effects on thyroid hormone levels in vivo. Archives of toxicology 1991, 65, (1), 15-9. 52. Sjöden, P.-O.; Söderberg, U., Long-lasting effects of prenatal 2, 4, 5trichlorophenoxyacetic acid on open-field behavior in rats: Pre- and postnatal mediation. Physiological Psychology 1975, 3, (2), 175-178. 53. Wojtczak, A.; Cody, V.; Luft, J. R.; Pangborn, W., Structures of human transthyretin complexed with thyroxine at 2.0 A resolution and 3',5'-dinitro-N-acetyl-L-thyronine at 2.2 A resolution. Acta crystallographica. Section D, Biological crystallography 1996, 52, (Pt 4), 75865. 54. Rice, P.; Longden, I.; Bleasby, A., EMBOSS: the European Molecular Biology Open Software Suite. Trends in genetics : TIG 2000, 16, (6), 276-7. 55. Schrödinger Multiple Sequence Viewer, Schrodinger, LLC, New York, NY., 2017. 56. Ferguson, R. N.; Edelhoch, H.; Saroff, H. A.; Robbins, J.; Cahnmann, H. J., Negative cooperativity in the binding of thyroxine to human serum prealbumin. Preparation of tritiumlabeled 8-anilino-1-naphthalenesulfonic acid. Biochemistry 1975, 14, (2), 282-9. 57. Cheng, S. Y.; Pages, R. A.; Saroff, H. A.; Edelhoch, H.; Robbins, J., Analysis of thyroid hormone binding to human serum prealbumin by 8-anilinonaphthalene-1-sulfonate fluorescence. Biochemistry 1977, 16, (16), 3707-13. 58. Kabsch, W., Xds. Acta crystallographica. Section D, Biological crystallography 2010, 66, (Pt 2), 125-32. 59. Winn, M. D.; Ballard, C. C.; Cowtan, K. D.; Dodson, E. J.; Emsley, P.; Evans, P. R.; Keegan, R. M.; Krissinel, E. B.; Leslie, A. G.; McCoy, A.; McNicholas, S. J.; Murshudov, G. N.; Pannu, N. S.; Potterton, E. A.; Powell, H. R.; Read, R. J.; Vagin, A.; Wilson, K. S., Overview of the CCP4 suite and current developments. Acta crystallographica. Section D, Biological crystallography 2011, 67, (Pt 4), 235-42. 60. McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R. J., Phaser crystallographic software. Journal of applied crystallography 2007, 40, (Pt 4), 658-674. 61. Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L. W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H., PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta crystallographica. Section D, Biological crystallography 2010, 66, (Pt 2), 213-21. 62. Emsley, P.; Cowtan, K., Coot: model-building tools for molecular graphics. Acta crystallographica. Section D, Biological crystallography 2004, 60, (Pt 12 Pt 1), 2126-32.


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63. McNicholas, S.; Potterton, E.; Wilson, K. S.; Noble, M. E., Presenting your structures: the CCP4mg molecular-graphics software. Acta crystallographica. Section D, Biological crystallography 2011, 67, (Pt 4), 386-94. 64. Case, D. A.; Darden, T. A.; Gusarov, S.; Kovalenko, A.; Kollman, P. A., Amber 2015 (AmberTools15 and Amber14), University of California, San Francisco. 2015. 65. Hou, T.; Wang, J.; Li, Y.; Wang, W., Assessing the performance of the MM/PBSA and MM/GBSA methods. 1. The accuracy of binding free energy calculations based on molecular dynamics simulations. Journal of chemical information and modeling 2011, 51, (1), 69-82. 66. Morgado, I.; Melo, E. P.; Lundberg, E.; Estrela, N. L.; Sauer-Eriksson, A. E.; Power, D. M., Hormone affinity and fibril formation of piscine transthyretin: the role of the N-terminal. Mol Cell Endocrinol 2008, 295, (1-2), 48-58. 67. Chen, Q.; Wang, X.; Shi, W.; Yu, H.; Zhang, X.; Giesy, J. P., Identification of Thyroid Hormone Disruptors among HO-PBDEs: In Vitro Investigations and Coregulator Involved Simulations. Environmental science & technology 2016, 50, (22), 12429-12438. 68. Meerts, I. A.; van Zanden, J. J.; Luijks, E. A.; van Leeuwen-Bol, I.; Marsh, G.; Jakobsson, E.; Bergman, A.; Brouwer, A., Potent competitive interactions of some brominated flame retardants and related compounds with human transthyretin in vitro. Toxicological sciences : an official journal of the Society of Toxicology 2000, 56, (1), 95-104. 69. Eneqvist, T.; Lundberg, E.; Nilsson, L.; Abagyan, R.; Sauer-Eriksson, A. E., The transthyretin-related protein family. European journal of biochemistry 2003, 270, (3), 518-32. 70. Iakovleva, I.; Begum, A.; Pokrzywa, M.; Walfridsson, M.; Sauer-Eriksson, A. E.; Olofsson, A., The flavonoid luteolin, but not luteolin-7-O-glucoside, prevents a transthyretin mediated toxic response. PloS one 2015, 10, (5), e0128222. 71. Power, D. M.; Llewellyn, L.; Faustino, M.; Nowell, M. A.; Bjornsson, B. T.; Einarsdottir, I. E.; Canario, A. V.; Sweeney, G. E., Thyroid hormones in growth and development of fish. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 2001, 130, (4), 447-59. 72. Ghosh, M.; Meerts, I. A.; Cook, A.; Bergman, A.; Brouwer, A.; Johnson, L. N., Structure of human transthyretin complexed with bromophenols: a new mode of binding. Acta crystallographica. Section D, Biological crystallography 2000, 56, (Pt 9), 1085-95. 73. Lans, M. C.; Klasson-Wehler, E.; Willemsen, M.; Meussen, E.; Safe, S.; Brouwer, A., Structure-dependent, competitive interaction of hydroxy-polychlorobiphenyls, -dibenzo-pdioxins and -dibenzofurans with human transthyretin. Chemico-biological interactions 1993, 88, (1), 7-21. 74. Weiss, J. M.; Andersson, P. L.; Zhang, J.; Simon, E.; Leonards, P. E.; Hamers, T.; Lamoree, M. H., Tracing thyroid hormone-disrupting compounds: database compilation and structure-activity evaluation for an effect-directed analysis of sediment. Analytical and bioanalytical chemistry 2015, 407, (19), 5625-34. 75. Montano, M.; Cocco, E.; Guignard, C.; Marsh, G.; Hoffmann, L.; Bergman, A.; Gutleb, A. C.; Murk, A. J., New approaches to assess the transthyretin binding capacity of bioactivated thyroid hormone disruptors. Toxicological sciences : an official journal of the Society of Toxicology 2012, 130, (1), 94-105. 76. Gunnarsson, L.; Jauhiainen, A.; Kristiansson, E.; Nerman, O.; Larsson, D. G., Evolutionary conservation of human drug targets in organisms used for environmental risk assessments. Environmental science & technology 2008, 42, (15), 5807-13.


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638 639 640 641 642 643 644

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77. Ucan-Marin, F.; Arukwe, A.; Mortensen, A.; Gabrielsen, G. W.; Fox, G. A.; Letcher, R. J., Recombinant transthyretin purification and competitive binding with organohalogen compounds in two gull species (Larus argentatus and Larus hyperboreus). Toxicol. Sci. 2009, 107, (2), 440-50. 78. Purkey, H. E.; Dorrell, M. I.; Kelly, J. W., Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma. Proceedings of the National Academy of Sciences of the United States of America 2001, 98, (10), 5566-71.

645 646 647 648 649


24

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650


GRAPHICAL ABSTRACT

651

652 653 654


25


Page 26 of 31

655

Table 1. Studied compounds with abbreviations, chemical abstract services (CAS) registry

656

numbers, usage, and binding affinities (Kd) determined using isothermal titration calorimetry. Compounds (Abbreviations)

CAS number

Use

3,3',5,5'-tetraiodo-Lthyronine (T4)

51-48-9

Fish

Human

Ratio

Kd (µM)

Kd (µM)

Fish:Human Kd

Control

0.23a (0.01b66)

0.09a (0.015b77) (0.084b78)

~1:1-1:3

2,2',4,4'131-55-5 tetrahydroxybenzophe none (BP2)

UV absorber

54.61

0.10

1:500

2-(3-chloro-217737-65-4 methylanilino)pyridin e-3-carboxylic acid (Clonixin)

Pharmaceutical

3.61

0.52

1:7

335-67-1

Surfactant

2.16

2.19

1:1

2,2',6,6'tetrabromobisphenol A (TBBPA)

79-94-7

Flame retardant

0.18

0.02

1:9

3,5,6-Trichloro-2pyridinol (TC2P)

6515-38-4

Herbicide

28.41

3.34

1:9

2,4,5trichlorophenoxyaceti c acid (2,4,5-T)

93-76-5

Herbicide

10.20

3.19

1:3

pentadecafluorooctan oic acid (PFOA)

657 658

a

The binding affinity of T4 is only an approximate value due to its thermoinstability and poor solubility in the isothermal titration calorimetry buffer. bKd values reported in literatures.

659


26

Page 27 of 31

660


FIGURE LEGENDS

661 662

Figure 1. Structural comparison of SaTTR (PDB code: 1SN0) and hTTR (PDB code: 2ROX). (a)

663

Ribbon drawing of the superimposed tetrameric structures, SaTTR in green and hTTR in gray.

664

SaTTR has a tetramer in the asymmetric unit; these are labelled A, B, C and D. hTTR has a

665

dimer in the asymmetric unit of the crystal; these are labelled A and B, and the symmetry-related

666

monomers in the hTTR tetramer are consequently labelled A’ and B’. T4 bound at the two

667

thyroxine binding sites are shown as sticks. (b) Close-up view of the thyroid-binding sites of

668

monomers B and B´ in hTTR and monomers B and D in SaTTR. Note that T4 in hTTR

669

penetrates deeper into the thyroid-binding site than the T4 in SaTTR. c) Global pair-wise

670

sequence alignment of SaTTR and hTTR. Residues constituting the thyroid-binding site are

671

highlighted in red.

672

Figure 2. Compound binding in the thyroid-binding site formed by monomers B and D in SaTTR

673

and monomers B and B´ in hTTR; (a) SaTTR-BP2, (b) SaTTR-clonixin (c) SaTTR-PFOA (d)

674

SaTTR-TBBPA (e) SaTTR-TC2P, (f) hTTR-TC2P, (g) SaTTR-2,4,5-T, (h) hTTR-2,4,5-T. The

675

refined (2m|Fo|−D|Fc|) electron density is shown in gray mesh at 1σ contour level. Water

676

molecules around the ligands are shown as red spheres.

677

Figure 3. Superimposed structures of SaTTR (green) and hTTR (gray) in complex with: (a) BP2

678

(hTTR-BP2, PDB code: 5JIQ), (b) clonixin (hTTR-clonixin, PDB code: 5L4I), (c) PFOA

679

(hTTR-PFOA, PDB code: 5JID), (d) TBBPA (hTTR-clonixin, PDB code: 5HJG), (e) TC2P, and

680

(f) 2,4,5-T. Selected water molecules are shown as red spheres. Hydrogen bonds are shown as

681

dashed lines.


27


Figure1 349x240mm (96 x 96 DPI)


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TOC 202x83mm (96 x 96 DPI)


Interspecies Variation Between Fish and Human Transthyretins in

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