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Originally published In Press as doi:10.1074/jbc.M305725200 on July 7, 2003

J. Biol. Chem., Vol. 278, Issue 38, 36887-36896, September 19, 2003
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The Iodothyronine Selenodeiodinases Are Thioredoxin-fold Family Proteins Containing a Glycoside Hydrolase Clan GH-A-like Structure*

Isabelle Callebaut {ddagger}, Cyntia Curcio-Morelli §, Jean-P. Mornon {ddagger}, Balazs Gereben  ||, Christoph Buettner §, Stephen Huang §, Bertrand Castro {ddagger} **, Tatiana L. Fonseca §, John W. Harney §, P. Reed Larsen § and Antonio C. Bianco § {ddagger}{ddagger}

From the {ddagger}Poôle Bio, Laboratoive de Minéralogie-Cristallographie de Paris, CNRS UMR7590, Universities Paris 6 and Paris 7, Paris 75252 Cedex 05, France, ||Department of Endocrine and Behavioral Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest H-1083 Hungary, §Thyroid Section, Division of Endocrinology, Diabetes and Hypertension, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, and **Sanofi-Synthelabo, 94255 Gentilly Cedex, France

Received for publication, June 2, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The three iodothyronine selenodeiodinases catalyze the initiation and termination of thyroid hormone effects in vertebrates. Structural analyses of these proteins have been hindered by their integral membrane nature and the inefficient eukaryotic-specific pathway for selenoprotein synthesis. Hydrophobic cluster analysis used in combination with Position-specific Iterated BLAST reveals that their extramembrane portion belongs to the thioredoxin-fold superfamily for which experimental structure information exists. Moreover, a large deiodinase region imbedded in the thioredoxin fold shares strong similarities with the active site of iduronidase, a member of the clan GH-A-fold of glycoside hydrolases. This model can explain a number of results from previous mutagenesis analyses and permits new verifiable insights into the structural and functional properties of these enzymes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The main secretory product of the thyroid gland is a prohormone thyroxine (T4), which must be monodeiodinated to 3,3',5-triiodothyronine (T3) by removal of an outer ring iodine to permit its binding to nuclear T3 receptors. These ligand-dependent transcription factors regulate genes critical for normal growth, central nervous system development, and energy homeostasis in all vertebrates (1). The specific monodeiodination of T4 in the outer ring is catalyzed by the types 1 or 2 iodothyronine selenodeiodinases (D1 or D2). Termination or prevention of thyroid hormone action is controlled by the inner ring deiodination of T3 or T4, respectively, catalyzed by a third deiodinase, D3. Thus, specific iodothyronine monodeiodinations are critical steps in both the activation and inactivation of thyroid hormones (2). The complex regulation of the activities of these selenocysteine (Sec)-containing enzymes permits modulation of T3 concentrations in specific cells controlling processes diverse as metamorphosis and adaptive thermogenesis. In adult vertebrates, the role of the deiodinases is primarily homeostatic, adjusting T3 production in response to environmental stresses such as iodine deficiency, starvation, or thermal challenges. In addition, the rapid conversion of T4 to T3 by D2 in the central nervous system and pituitary permits accurate monitoring of circulating T4, allowing the feedback regulation of thyroid-stimulating hormone secretion based in part upon circulating pro-hormone (T4) concentrations (2).

Although there are important differences among the three deiodinases with respect to their catalytic functions, they have notable similarities. All are integral membrane proteins of 29-33 kDa and have regions of high similarity in the area surrounding the active center Sec, the critical residue that confers deiodinases with high catalytic activity (3-5). Although there is some structure-function information available, particularly for D1, our understanding of the catalytic mechanisms and three-dimensional conformation of these proteins is limited because of the inability to synthesize large quantities of soluble, catalytically active proteins for crystallization purposes. This is a result both of the apparent importance of the transmembrane (TM) domain as well as the major differences in the pathways for selenoprotein synthesis between prokaryotic and eukaryotic organisms (2, 6).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Strategy—Overlap extension PCR or restriction digestion were used to create single or multiple mutations in the deiodinase structures to test the reliability of our predicted three-dimensional model of the active center (Fig. 4). The choice of amino acids was made based on the chemical conservation of several positions within the whole deiodinase family with respect to the conserved features of the thioredoxin (TRX) and GH templates. The mutations were made on wild type deiodinase sequences fused to a FLAG peptide in the NH2 terminus, previously shown not to interfere with catalysis (7). These constructs and the corresponding wild type versions of each deiodinase were transiently expressed in human embryonic kidney (HEK-293) epithelial cells and assayed for activity. Immunoprecipitation with anti-FLAG antibody allowed us to quantify 35S metabolically labeled deiodinases.



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  		FIG. 4.
Schematic representation of the putative active site of deiodinases deduced from sequence alignment (Fig. 1) and from the associated modeling (Fig. 2). The positions shown are those of D2, and the table contains the corresponding positions and residues in D1 and D3. According to this model, H-bonds/ion pairs between His-165 and the carboxyl group stabilize the iodothyronine. A similar interaction might take place between the hydroxyl group and Ser-128 in D1 but not in D2 and D3 where a Pro (P135) replaces Ser. Assuming these anchors, the essential Sec133 lies between the inner and outer rings of the iodothyronine where it might interact with iodine atoms. The specificity of D1, D2, and D3 is further tuned by several amino acids in close proximity, e.g. Ser-130, Asp-162, and Ser-167). Two other groups of amino acids centered on the conserved Glu-228/Phe-128 and Trp-170/His-185 positions are in the vicinity of the active center and modulate its functional properties. After His-165, the IDUA-like IOD insertion has a speculative character, and alternatively, the highly conserved Trp-170 may directly interact with T4 molecules as well as His-185. The IDUA-like IOD insertion probably constitutes a cap that may cover the active site upon ligand binding.

 



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  		FIG. 1.
Sequence alignment of the three deiodinases with three membranous members of the TRX superfamily. This alignment was built manually on the basis of Hydrophobic Cluster Analysis (see "Results and Discussion"). The top panel gathers the three deiodinase sequences (D1, D2, and D3) from human (Hs) (Swiss-Protein Data Bank (SW) accession numbers P49895 [GenBank] , Q92813 [GenBank] , and P55073 [GenBank] , respectively). The three members of the TRX superfamily (GPX) family following the structural classification of proteins classification (19)) are the thiol:disulfide interchange protein from D. radiodurans (Dr) DR0345 (GenBankTM identification (gi) number 7471775), Bradyrhizobium japonicum (Bj) TlpA (SW: P43221 [GenBank] ) and B. japonicum (Bj) CcmG/DsbE (SW: P30960 [GenBank] ). The nonmembranous parts of the two latter are known at the three-dimensional level (Protein Data Bank identifiers 1JFU [PDB] and 1KNG [PDB] , respectively). The observed secondary structures are shown below the alignment ({beta}-strands are symbolized by arrows). The vertical pink and purple arrows indicate the limits of the reported atomic coordinates. Sequences of four canonical examples of the TRX-fold {beta}{alpha}{beta} motif are also shown (Arabidopsis thaliana (At) and Brassica napus (Bn) TRX H-type 2 THH2 (SW: Q38879 [GenBank] and Q39362 [GenBank] , respectively); human (Hs) TRX (SW P10599 [GenBank] ) and Bos taurus gluthatione peroxidase (GPX, SW: P00345 [GenBank] )). The secondary structures of human TRX and GPX (Protein Data Bank codes 1AUC [PDB] and 1GP1 [PDB] , respectively) are also indicated below the sequences. A segment of the human iduronidase sequence (IDUA, SW: P35475 [GenBank] ), which shares strong local similarities with the deiodinase active site insertion within the TRX scaffold, is shown above the alignment along the {beta}{alpha}{beta} motif and the IDUA-like IOD active-site insertion. This last region, the three-dimensional structure of which is not known, is aligned with a segment of the Fusarium moniliforme endopolygalacturonase (Protein Data Bank code 1HG8 [PDB] ) and of the Bacillus subtilis subtilisin (Protein Data Bank code 1SCJ [PDB] ), both structures that may constitute local three-dimensional templates for modeling the IDUA-like IOD insertion (see Fig. 2). The position of the putative TM segment is shaded light yellow with polar amino acids in critical positions highlighted (blue = basic, red = acidic, green = hydrophobic). H indicates a probable hinge linking the TM to the globular domain. The main probable "topohydrophobic" positions (positions which are always occupied by hydrophobic amino acids, and which are crucial for the fold) are indicated with gray circles above the alignment and positions of the mutations reported in this study are indicated by red stars. The essential Sec (X) or Cys (C) are reported on an orange background. Noticeable identities or similarities are shown on black and gray backgrounds, respectively. Sequence numbering is indicated at right. Black dots represent the COOH termini. Numbers within brackets indicate the lengths of segments that are not shown. This alignment was quantitatively assessed on 133 aligned positions within the secondary structure cores. The mean pair wise sequence identity between deiodinases and GPX-like proteins is 19% (25% between D3 and Dr0345 with a significant Z-score of 10 {sigma} for 1,000 random alignments). Similar significant Z-scores are also found considering the similarity index using the Blosum62 matrix or the HCA score (percent of positions where a strong hydrophobicity is present in both the compared sequences). Note: reinforcing the relationship between deiodinases and the TRX fold, we also note a striking sequence similarity in the NH2 terminus between D2 {beta}C(SSVVHVSSTE) and {beta}0 of the TRX2 from Dictyostelium discoideum (SW: P29446 [GenBank] ) (SRVIHISSNE).

 


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  		FIG. 2.
Three-dimensional structures and secondary structure organizations of the archetype TRX enzyme (Protein Data Bank code 1AUC [PDB] ) (A), B. japonicum CcmG (Protein Data Bank code 1KNG [PDB] ) (B), B. japonicum TlpA (Protein Data Bank code 1JFU [PDB] ) (C) and of the overall rough model of the D2 deiodinase (amino acid Leu-77 to the COOH-terminal Arg-265) (D), deduced from the HCA-based alignment shown in Fig. 1. Colors and labels are as in Fig. 1. N and C indicate NH2 and COOH termini, respectively (N' when only a relative one). The deiodinase model was built and roughly refined using the SwissPDBViewer tool (41) using TlpA as template with the exception of {alpha}2 and {alpha}3 that were modeled on the basis of the CcmG template exhibiting a local better sequence similarity with D2 (see Fig. 1). No nonsolvable inconsistencies have been observed, and all of the TRX-fold elements are well defined. Several segments were not modeled: NH2-terminal to L76 where the TM segment lies; the long D2 hydrophilic insertion T92 to G109 within the {beta}C-{beta}D loop ({Delta}D2); and the small SSLS insertion within the IDUA-like IOD active-site insertion between {beta}2 (yellow) and {alpha}B (white). The active-site insertion, which is likely to function as a cap on the active site, was tentatively modeled on the template of two segments belonging to B. subtilis subtilisin (Protein Data Bank code 1SCJ [PDB] ) for the first part (blue helix) and to Fusarium moniliforme endopolygalacturonase (Protein Data Bank code 1HG8 [PDB] ) for the segment returning to {alpha}B. These two templates have been selected for their local sequence similarities with deiodinases and for their compatible three-dimensional paths with the main part of the model. The relative Ala-rich content of this segment in deiodinases weight up the W, F, V, and T propensities for extended structures and may favor the formation of a helical path as shown here. The essential Cys or Sec are shown as yellow and orange balls, respectively. T4 is shown in green at an approximate localization within the active site region in which Glu-163 (red) and His-165 (blue) are reported.

 
Constructs—The constructs CC31, BG121, and CC32 were described previously (7). CC31 is an NH2 terminus FLAG-tagged wild type human D1 (FLAG-wtD1). BG121 is a COOH terminus FLAG-tagged wild type human D2 (FLAG-wtD2), and CC32 is an NH2 terminus FLAG-tagged wild type human D3 (FLAG-wtD3).

BG159 is a human D1 in which Pro replaced Ser-128 (S128P D1). Using overlap extension PCR, the fragments obtained with the oligonucleotides Bp169-Bp171 (Bp169 sense, 5'-GGAATTCATTATGGGGCTGCCCCAGCCAGGGCT-3', and Bp171 antisense, 5'-TTGAACATAAATGGAGGTCAGGTACA-3') and Bp169/C53 (Bp170 sense, 5'-TGTACCTGACCTCCATTTATGTTCAA-3', and C53 antisense (7)) and hD1 template (8) were used as template for another PCR using only Bp169-C53. The resulting fragment was cut by EcoRI/HindIII and cloned into EcoRI/HindIII sites of a D10 vector containing a minimal SECIS element.

CC29 is an NH2 terminus FLAG-tagged human D1 in which an Ala replaced the Glu-156 (E156A D1). Using overlap extension PCR, the fragments obtained with the oligonucleotides C52/C44 (C52 sense (7) and C44 antisense, 5'-AGCCATCTGATGCATGTGCT GCTTCAATGTAAATGACAAGAAAATCT-3') and C43/C53 (C43 sense, 5'-AGATTTTCTTGTCATTTACATTGAAGCAGCACATGCATCAGATGGCT-3' and C53 antisense (7)) and CC31 as template were used as a template for another PCR reaction using only the outer oligonucleotides C52/C53 and the resulting fragment was subcloned in EcoRI/HindIII sites of CC31.

CC34 is an NH2 terminus FLAG-tagged human D1 in which an Asp replaced Glu (E156D D1). Using overlap extension PCR, the fragments obtained with the oligonucleotides C52/C56 (C56 antisense, 5'-AGCCATCTGATGCATGTGCGTCTTCAATGTAAATGACAAGAAAATCT-3') and C54/C53 (C54 sense, 5'-AGATTTTCTTGTCATTTACATTGAAGACGCACATGCATCAGATGGCT-3') and CC31 as template were used as a template for another PCR reaction using only the outer oligonucleotides C52/C53. The resulting fragment was subcloned in EcoRI/HindIII sites of CC31.

CC36 is an NH2 terminus FLAG-tagged human D1 in which an Ala replaced Trp 163 (W163A D1). Using overlap extension PCR, the fragments obtained with the oligonucleotides C52/C5 (C63 antisense, 5'-GTTCTTAAAAGCCGCGCCATCTGATGC-3') and C62/C53 (C62 sense, 5'-GCATCAGATGGCGCGGCTTTTAAGAAC-3') and CC31 as template were used as a template for another PCR reaction using only the outer oligonucleotides C52/C53. The resulting fragment was subcloned in EcoRI/HindIII sites of CC31.

CC35 is an NH2 terminus FLAG-tagged human D1 in which an Ala replaced Glu (E214A D1). Using overlap extension PCR, the fragments obtained with the oligonucleotides C52/C58 (C58 antisense, 5'-TACGCAGCACTGCCTGCGAGGCTCTACATA-3') and C59/C53 (C59 sense, 5'-TATGTAGAGCCTCGCAGGCAGTGCTGCGTA-3') and CC31 as template were used as a template for another PCR reaction using only the outer oligonucleotides C52/C53. The resulting fragment was subcloned in EcoRI/HindIII sites of CC31.

In the F128A D2 protein, an Ala replaces Phe-128. Using overlap extension PCR, the fragments obtained with the oligonucleotides Bp97/A2 (A2 antisense, 5'-AGTGGCTGAGCCAGCGTTGACCACTAG-3') and A1/A8 (A1sense, 5'-GATCACCAGTTGCGACCGAGTCGGTGA-3', and A8 antisense, 5'-TAAACCAGCTAATCTAGTTTTCTTTCATCTCTT-3') and hD2Selp (9) as template were used as a template for another PCR reaction using only the outer oligonucleotides Bp97/A8. The resulting fragment was subcloned in EcoRI/PstI sites of hD2Selp.

In the P134A D2 protein, an Ala replaces Pro-134. Using overlap extension PCR, the fragments obtained with the oligonucleotides Bp97/A4 (A4 antisense, 5'-CTGGCTCGTGAAAGGAGCTCAAGTGGCTGAGCC-3') and A3/A8 (A3sense, 5'-GGCTCAGCCACTTGAGCTCCTTTCACGAGCCAG-3') and hD2Selp (9) as template were used as a template for another PCR reaction using only the outer oligonucleotides Bp97/A8. The resulting fragment was subcloned in EcoRI/PstI sites of hD2Selp.

In the P135S D2 protein, a Ser replaces Pro-135. Using overlap extension PCR, the fragments obtained with the oligonucleotides Bp97/A6 (A6 antisense, 5'-CAGCTGGCTCGTGAAAGAAGGTCAAGTGGC-3') and A5/A8 (A5sense, 5'-GCCACTTGACCTTCTTTCACGAGCCAGCTG-3') and hD2Selp (9) as template were used as a template for another PCR reaction using only the outer oligonucleotides Bp97/A8. The resulting fragment was subcloned in EcoRI/PstI sites of hD2Selp.

CC39 is COOH terminus FLAG-tagged human D2 in which Asp replaced Glu-163 (E163D D2). Using overlap extension PCR, the fragments obtained with the oligonucleotides Bp97/C71 (C71 antisense, 5'-TGATGGATGAGCGTCATCAATGTAGAC-3') and C70/Bp85 (C70 sense, 5'-GTCTACATTGATGACGCTCATCCATCA-3') and BG121 as template were used as a template for another PCR reaction using only the outer oligonucleotides Bp97/Bp85 and the resulting fragment was subcloned in EcoRI/ApaI sites of Bg121.

CC25 is COOH terminus FLAG-tagged human D2 in which Ala replaced Glu-163 (E163A D2). Using overlap extension PCR, the fragments obtained with the oligonucleotides Bp97/C40 (Bp97 sense, 5'-GGAATTCATTATGGGCATCCTCAGCGTAGACTTGCTGATCA-3', and C40 antisense, 5'-ATCTGATGGATGAGCCGCATCAATGTAGACCAGCAG-3') and C39/Bp85 (C39 sense, 5'-CTGCTGGTCTACATTGATGCGGCTCATCCATCAGAT-3' and Bp85 antisense, 5'-TTCCGCGGCCGCTATGGCCGACGTCGACTTAACCAGCTAATCTAGTTTTCTTTCATCT-3') and BG121 as template were used as a template for another PCR reaction using only the outer oligonucleotides Bp97/Bp85 and the resulting fragment was subcloned in EcoRI/ApaI sites of Bg121.

CC38 is COOH terminus FLAG-tagged human D2 in which Asn replaced His-165 (H165N D2). Using overlap extension PCR, the fragments obtained with the oligonucleotides Bp97/C67 (C67 antisense, 5'-AGCCATCTGATGGATTAGCCTCATCAAT-3') and C/Bp85 (C66 sense, 5'-ATTGATGAGGCTAATCCATCAGATGGCT-3') and BG121 as template were used as a template for another PCR reaction using only the outer oligonucleotides Bp97/Bp85 and the resulting fragment was subcloned in EcoRI/ApaI sites of Bg121.

CC37 is COOH terminus FLAG-tagged human D2 in which Ala replaced Trp-170 (W170A D2). Using overlap extension PCR, the fragments obtained with the oligonucleotides Bp97/C65 (C65 antisense, 5'-GTCCCCCGGTATCGCCGCGCCATCTGATGGATG-3') and C/Bp85 (C64 sense, 5'-CATCCATCAGATGGCGCGGCGATACCGGGGGAC-3') and BG121 as template were used as a template for another PCR reaction using only the outer oligonucleotides Bp97/Bp85 and the resulting fragment was subcloned in EcoRI/ApaI sites of Bg121.

CC40 is COOH terminus FLAG-tagged human D2 in which Gln replaced His-185 (H185Q D2). Using overlap extension PCR, the fragments obtained with the oligonucleotides Bp97/C73 (C73 antisense, 5'-TCCTGGTTCTGCTGCTTCTTCACCTCAAAAG-3') and C72/Bp85 (C72 sense, 5'-CTTTTGAGGTGAAGAAGCAGCAGAACCAGGA-3') and BG121 as template were used as a template for another PCR reaction using only the outer oligonucleotides Bp97/Bp85 and the resulting fragment was subcloned in EcoRI/ApaI sites of Bg121.

BG158 is a human D3 in which a Ser replaced the Pro-146 (P146S D3). Using overlap extension PCR, the fragments obtained with the oligonucleotides Bp78-Bp168 (Bp78 sense (7) and Bp168 antisense, 5'-GCGCCATGAACGATGGTCAGGTGCAGCT-3') and Bp167/162 (Bp167 sense, 5'-TGCACCTGACCATCGTTCATGGCGCGCA-3', and Bp162 antisense, 5'-CCCAAGCTTGGGTTACACCCTCCGGGGCCGAGCGCCGT-3') and hD3 CDM8 template (10) were used as template for another PCR using only Bp78-162. The resulting fragment was cut by EcoRI/HindIII and cloned into EcoRI/HindIII sites of a D10 vector containing a minimal SECIS element.

CC30 is an NH2 terminus FLAG-tagged human D3 in which Ala replaced Glu-174 (E174A D3). Using overlap extension PCR, the fragments obtained with the oligonucleotides C50/C48 (C50 sense (7) and C48 antisense, 5'-TCGGAGGGGTGCGCTGCCTCGATGTAGATGATGAG-3') and C47/C51 (C47 sense, 5'-CTCATCATCTACATCGAGGCAGCGCACCCCTCCGA-3', and C51 antisense (7)) and CC32 as template were used as a template for another PCR reaction using only the outer oligonucleotides C50/C51 and the resulting fragment was subcloned in EcoRI/HindIII sites of CC32.

Deiodinase Assays—D1, D2, and D3 were assayed as described previously (7, 11) under specific conditions as indicated under "Results and Discussion." Metabolic labeling with 35S or 75Se followed by immunoprecipitation in sonicates of cells transiently expressing deiodinases was done as described by Baqui et al. (12).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The Selenodeiodinases Contain a TRX Fold—To gain further insights into the structures of these proteins, we used hydrophobic cluster analysis (HCA)1 (13, 14), a sensitive method based among others on the fundamental principles of protein fold and on a two-dimensional transposition of sequences (see Refs. 15 and 16 for examples on enzyme families), allowing the resolution of a sequence into its regular secondary structures centered on the so-defined hydrophobic clusters. The three deiodinase proteins (D1, D2, and D3) show considerable similarity (~ 50% sequence identity, Fig. 1). Their general structure indicates a single TM segment, which is present in the NH2 termini of D1, D2, and D3 (Fig. 1, shaded light yellow), whereas several clusters typical of {alpha}-helices or {beta}-strands are found well conserved, suggesting that they could correspond to core secondary structures of the deiodinase globular domains. Loops of variable lengths can be identified between conserved clusters. The first large one (Fig. 1, designated H) probably corresponds to a hinge separating the predicted TM segment from the globular domain.

We used deiodinase sequences as queries in PSI-BLAST searches (17) (default values, nonredundant (NR, 1,027,609 sequences), and Swiss-Protein (SW 100,395 sequences) databases at NCBI). Just below the significance threshold value (expected E-value > 10-3), we observed similarities with various members of the TRX or TRX-like families. The first hit of a search against the NR data base using the D1 sequence as query was the thiol:disulfide interchange protein from Deinococcus radiodurans (DR0345, E-value of 0.95; 27% identity over the 84 amino acids 86-169 of D1), whereas the first hit of a similar search against the Swiss-Protein data base with the D2 sequence was the TRX-H-type 2 (TRX-H2) and TRX-H-type 4 (TRX-H4) from Arabidopsis thaliana and Brassica napus, respectively (E-value of 0.36 and 0.38, respectively; 37% over the 40 amino acids 124-163 of D2). These similarities are centered on the deiodinase Sec-containing active center (Fig. 1, X, shaded orange), which is aligned with the first of the active-site Cys of TRXs. In the TRX-fold family, not all of the members have a redox-active disulfide. Instead, the glutathione peroxidase (GPX) indeed has a Sec that interacts with glutathione substrate, which can be aligned with the accessible Cys of the redox protein (the first Cys of the couple) (Fig. 1) (18). Within the TRX-fold family, the deiodinase can thus also be compared with GPX, both having a Sec that can be aligned with the first active Cys of the TRX redox couple. Both are also characterized by a ping-pong reaction mechanism with a specific first substrate (R-OH or iodothyronine) and a thiol second substrate.

The observed similarities with TRX and TRX-like proteins were further strengthened at a two-dimensional level using HCA as they are associated with a predicted conservation of structural features (Fig. 1). In both families of proteins, the "active" Cys or Sec is located in a loop separating a {beta}-strand and an {alpha}-helix corresponding to the {beta}1/{alpha}1-structures of the TRX fold (Fig. 1, red and orange). The {beta}-strand COOH-terminal to the {alpha}-helix (Fig. 1, strand {beta}2 of the TRX fold, yellow) is also well conserved, clearly indicating that the deiodinase region encompassing amino acids 115-156 (D1 numbering) corresponds to the {beta}{alpha}{beta} motif of the TRX fold (Fig. 1, boxed). This hypothesis was further assessed by threading procedures, which significantly detected proteins of the GPX-like family within the structural classification of proteins (19). TRX-like fold superfamily (the structures of the thiol:disulfide interchange proteins CcmG/DsbE (Protein Data Bank code 1kng [PDB] ) and TlpA (1jfu [PDB] ) and of tryparedoxin-i (1qk8 [PDB] ) were scored by three-dimensional position-specific scoring matrix (20) with E-values of 0.0883, 0.0174, and 0.0976, respectively. Those of peroxiredoxin 5 (1hd2 [PDB] and 1h4o [PDB] ) and TlpA (1jfu [PDB] ) were detected by FUGUE (21) with Z-scores of 8.63, 8.41, and 7.51, respectively.

However, no similarity could be found by PSI-BLAST with TRX-fold proteins downstream of the {beta}{alpha}{beta} motif, suggesting that the COOH-terminal part of deiodinases is different from that of the TRX fold or that extra elements, which do not belong to the canonical TRX fold, locally interrupt the relationship that could be established with it. The latter hypothesis is supported by the fact that a {beta}{beta}{alpha} motif (Fig. 1, {beta}3-{beta}4-{alpha}3) is predicted at the end of the deiodinase sequences that might thus correspond to the {beta}{beta}{alpha} motif of the COOH-terminal part of the TRX fold. The intervening sequences including amino acids between 156 and 210 of deiodinase (D1 numbering) thus might correspond to distinct secondary structure elements added to the canonical TRX-fold core as observed in DsbA or in the GPX-like family, all of which are members of the TRX fold that have inserted residues forming separate domains or winding around the TRX fold (18). This hypothesis was further supported by the threading results in which significant alignments of the COOH-terminal {beta}{beta}{alpha} motifs were obtained with the COOH-terminal sequences of deiodinases. This structural correspondence is clearly documented by the similarities observed between the deiodinase sequences and proteins of the GPX-like family (Fig. 1).

From the PSI-BLAST and threading results, we observed that the similarities between deiodinases and proteins of the GPX-like family (19) to which the structurally characterized CcmG (22) and TlpA (23) belong are not limited to the TRX {beta}{alpha}{beta} and {beta}{beta}{alpha} motifs. These similarities are well supported and refined using HCA at the two-dimensional level, and interestingly, the strictly or highly conserved amino acids are located on positions that are also conserved in the whole family of thiol:disulfide interchange proteins (Fig. 1) that can therefore serve as reliable anchor points for the alignment. For example, one can note the strict conservation of an Ala-Pro motif (residues 92-93 in D1) preceding a cluster typical of a {beta}-strand ({beta}C), which is associated in CcmG and TlpA with an extended structure (strand {beta}B in Figs. 1 and 2). Thus, deiodinases are predicted to have an NH2-terminal sequence similar to the NH2-terminal insert of CcmG and TlpA with three {beta}-strands ({beta}B, {beta}C, and {beta}D), two of which contribute to the {beta}-sheet on the strand {beta}2-side (Fig. 2). The large insertion found in the D2 sequence (18 amino acids within brackets on Fig. 1) lies exposed in the loop linking strand {beta}C and {beta}D (Fig. 2, {Delta}D2) in a similar place as the additional {beta}-hairpin of CcmG (22) and is probably located close to the active site. The two structures of CcmG and TlpA differ by the presence of an additional helix in the very NH2 terminus of TlpA, which does not exist in CcmG (Figs. 1 and 2, {alpha}A). In deiodinases, the presence of a large hydrophobic cluster, predicted as a {alpha}-helix followed by a predicted short {beta}-strand just after the TM segment, suggests that the counterparts of the {alpha}A and {beta}A structures could also be present. Although no clear amino acid similarities could be found for these structures between deiodinases and TlpA, such a hypothesis is supported at the two-dimensional level by the HCA and three-dimensional PSSM results.

Selenodeiodinases and Iduronidases Share Common Structural Features within Their Respective Catalytic Regions— Thiol:disulfide interchange proteins have a second insertion between the two TRX {beta}{alpha}{beta} and {beta}{beta}{alpha} motifs relative to the TRX canonical fold. This insertion provides an additional {alpha}-helix (Fig. 2, {alpha}B, white) lining the active site and also contributes to the {beta}-sheet by adding one additional {beta}-strand (Fig. 2, strand {beta}E, pink). As deiodinases also have an insertion at the same place and as the presence of strands {beta}C and {beta}D in deiodinase should involve that of strand {beta}E to form a continuous {beta}-sheet, we hypothesize that the deiodinase insertion could also be similar to those found in the CcmG and TlpA structures. A putative alignment of strand {beta}E (pink) was found with the VVVDT cluster, strongly predicted as a {beta}-strand (amino acids 196-200 in D1), which is encircled by two clusters typical of helical structures aligned with the helices {alpha}B and {alpha}2 of the CcmG and TlpA structures (Fig. 1).

Altogether, the alignment shown in Fig. 1 among D1, D2, D3, and the three GPX-like proteins, Dr0345, TlpA, and CcmG, is quantitatively assessed by statistical data (see legend of Fig. 1). With regards to this alignment, the region between amino acids 157 and 181 (D1 numbering) may thus correspond to a deiodinase-specific insertion relative to the CcmG and TlpA structures. This specific sequence may be crucial for the deiodinase function as it is positioned at least partially near the active site centered around the essential Sec residue (Fig. 2).

This highly conserved, deiodinase-specific segment between amino acids 152 and 166 (D1 numbering) shares striking similarities with {alpha}-L-iduronidase (IDUA, 47% identity with D1 and D3, 60% with D2) as revealed by PSI-BLAST searches (Fig. 1, IDUA). Although marginal because of its small length (E-value of 53 with iduronidase from Canis familiaris when searching the Swiss-Protein data base with the D2 sequence as query), this similarity was further supported by HCA analysis, significantly extended along a full {beta}{alpha}{beta}{alpha}{beta} segment of nearly a hundred amino acids corresponding to the TRX-fold {beta}1-{alpha}1-{beta}2-{alpha}B-{beta}E motif and assessed by a clear structural relationship between the GPX-TTR fold and this enzyme family (Fig. 3). Lysosomal {alpha}-L-iduronidase, which cleaves {alpha}-linked iduronic acid residues from the nonreducing end of the glycosaminoglycans, heparan sulfate, and dermatan sulfate belongs to family 39 of glycoside hydrolases (GH) (24). This family is included in the larger clan GH-A whose different members share the same sugar retaining mechanism (retention of configuration of the anomeric carbon of the substrate) and the same structure (a ({beta}/{alpha})8 barrel in which the acid/base and the nucleophilic residues are located at the COOH-terminal end of strands {beta}4 and {beta}7, respectively) (15, 25). These similarities led us to align strand {beta}2 of the predicted TRX {beta}{alpha}{beta} motif of deiodinase with strand {beta}7 of the GH with the strict conservation of the Glu, which in iduronidase (Glu-299) acts as the nucleophilic residue (Figs. 1, 2, 3, shaded red) (26). In both the TRX and GH folds, this Glu is positioned at the end of a {beta}-strand included in a parallel mode in a {beta}-sheet architecture (Fig. 3).



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  		FIG. 3.
A, alignment of the TRX-like active site region {beta}1-{alpha}1-{beta}2-{alpha}B-{beta}E with part of the active site region of clan GH-A glycoside hydrolases. Human {alpha}-L-iduronidase (Hs IDUA; Swiss-Protein Data Bank (SW): P35475 [GenBank] ) and {beta}-xylosidase B from Caldicellulosiruptor saccharolyticus (Cs XYNB; SW:P23552) belong to family GH39, whereas endoglucanase 5A from Bacillus agaradhaerens (Ba Cel5A; SW: O85465 [GenBank] ) belongs to family GH5 (24). The aligned sequence of the GPX-like family Dr0345 protein from Deinococcus radiodurans (Dr DR0345; Fig. 1) is shown above the deiodinase sequences. Secondary structures of the TRX fold and of the Cel5A structure (Protein Data Bank code 4A3H [PDB] ) (42) are reported. The mean pair wise sequence identity between deiodinases and iduronidase (59 aligned positions of the secondary structure core) is 24% (25% between D2 and iduronidase with a Z-score of 6.1 {sigma} for 1,000 random alignments). B, structural superimposition of the {beta}1-{alpha}1-{beta}2-{alpha}B-{beta}E motif of the partially refined deiodinase 2 model (see Fig. 2) with the {beta}6-{alpha}6-{beta}7-{alpha}7-{beta}8 motif of the GH Cel5A structure. The observed position of the B. agaradhaerens Cel5A substrate (2',4' dinitrophelnyl-2-deoxy-2-fluro-B-D-cellobioside, Protein Data Bank code 4A3H [PDB] ) and the predicted approximate position of T4 are shown. The essential Glu-163 of D2 and the catalytic Glu-228 of Cel5A are shown red. Sec133 is reported yellow, and His-165 is blue. The superimposition led to mean root mean square between core regular secondary structures in the range of 3-4 Å. C, chemical structure of deiodinase and here-reported glycoside hydrolases substrates.

 

Thus, regarding the functional properties of this acidic amino acid in GH and the compatible supersecondary structure among deiodinases, TRX, and GH in this region (the {beta}{alpha}{beta} motif corresponding to {beta}1-{alpha}1-{beta}2 and {beta}6-{alpha}6-{beta}7 in TRX and iduronidase, respectively), it is possible that this Glu in deiodinase (Glu-156, Glu-163, and Glu-174 in D1, D2, and D3, respectively) plays a key role. The position of Glu-156, predicted as critical, is located in the {beta}{alpha}{beta} motif of the deiodinase model constructed on the basis of the TlpA structure (Fig. 2), clearly establishing the proximity of this amino acid to the predicted position of the Sec. This position also often corresponds to an acidic residue in TRX-fold proteins (Fig. 1). It is worth noting that the local similarity observed between deiodinases and iduronidase highlighted here is all the more striking as a great sequence divergence is generally observed in the GH group, which can be aligned only with difficulty outside the core segments. The level of similarities between deiodinases and iduronidase is thus comparable to (or even better than) the mean level of similarities generally observed among GH proteins. Indeed, on the same 59 aligned positions as reported above, iduronidase and xylosidase (IDUA and XYNB in Fig. 3, both belonging to clan GH-A family 39) share only 15% sequence identity (Z-score of 3.1 {sigma}). Putatively, this local structural mimicry between deiodinases and iduronidase may rely partly on the overall similarity of their substrates, T4 (or T3) and sulfated {alpha}-L-iduronic acid, respectively, both based on O-linked hexagonal rings substituted by bulky groups ortho to the linker. These are not present to the same extent in xylan, the substrate of the more sequence-distant xylosidase (Fig. 3C). The substrate specificity could thus be determined in part by the IDUA-like deiodinase active-site insertion lying between strand {beta}2 ({beta}7 in GH proteins) and helix {alpha}B ({alpha}7), which is likely to act as a cap covering the active site.

These data are reminiscent of haloalkane dehalogenases, which belong to the family 6 of {alpha}-{beta} hydrolases, and in which a helical cap following a {beta}{alpha}{beta} motif with active site residues at the end of {beta} strands covers the ligand binding site (27). The IDUA-like insertion common to deiodinases and iduronidase is likely to play a similar role to that performed by these caps built on two successive {alpha}-hairpins based on the uteroglobin fold, a chlorinated biphenyl-binding protein (28). However, the IDUA-like insertion is predicted to consist of only one hairpin and thus should roughly correspond to one-half of an uteroglobin-like cap.

Site-directed Mutagenesis Confirms the Predicted Deiodinase Model and Provides Insights into the Catalytic Mechanisms for D1 and D2—The predicted structure of the three deiodinases, a single TM domain connected to a globular domain containing the catalytic center, is compatible with the predicted D1, D2, and D3 topology using protease protection assays, selective biotinylation, and immunofluorescence cytochemistry (12, 29, 30). The similarities of the structures of the three deiodinases would make it probable that variations of specific amino acids are responsible for the functional contrasts among D1, D2, and D3.

The model predicts that the active center of the three deiodinases is a Sec-containing pocket defined by the {beta}1-{alpha}1-{beta}2 motifs of the TRX fold and the IDUA-like insertion (Fig. 4). If this model is accurate, we would expect significant perturbations in enzyme function if critical amino acids in the binding pocket are changed, providing insights into the differences in iodothyronine deiodination catalyzed by D1, D2, and D3. D1 has a relatively low affinity for T4 (Km = 1-2 µM) and its catalytic activity is bi-substrate in nature with a thiol-containing cofactor serving as the second substrate with ping-pong kinetics, similar to GPX as mentioned earlier (31). D2 and D3, on the other hand, have relatively high affinity for T4/T3 (Km = 1-4 nM), and both exhibit sequential reaction kinetics, suggesting that the iodothyronine and the thiol-containing cofactor interact with the enzyme simultaneously before the reaction takes place (31). Interestingly, D1, D2, and D3 differ in their sensitivity to 6n-propyl-2-thiouracil (PTU). D1 is quite sensitive (Ki = 5 µM) but D2 and D3 are not (Ki >1 mM). It is assumed that PTU inhibits D1 by competing with the endogenous thiol-containing cofactor for a putative selenenyl iodide (E-Se-I) intermediate (31). Supporting this interpretation is the fact that PTU inhibition is uncompetitive with the first D1 substrate (iodothyronine) but competitive with the second, e.g. dithiothreitol (31). Because of the PTU insensitivity, it has been proposed that during D2 or D3-catalyzed deiodination, the leaving iodonium (I+) is abstracted by the endogenous cofactor, resulting in a enzyme-thyronine intermediate D2-T3 (or 3,3',5'-triiodothyronine, rT3) complex and a cofactor-S-I (32).

To test the model, our initial approach was to identify the critical residues in the putative active pocket that mediate the unique kinetic properties of each deiodinase by modifying the residues in the Thr-Sec-Pro-Pro/Ser-Phe in the transition between {beta}1 and {alpha}1 (Figs. 1 and 4). It is notable that this sequence is identical in D1, D2, and D3, only that in all of the D1 sequences with the exception of the PTU-insensitive D1 of the blue tilapia (Oreochromis aureus), the uncharged polar side chain Ser residue substitutes for the nonpolar side chain Pro at position 128 (33). We have focused our initial studies on D1 and D2 because there are more functional data available and these two enzymes catalyze T4 to T3 conversion.

Consistent with the expectations of the model, Ala replacement of the invariant Phe-136 inactivated D2, suggesting the occurrence of critical {pi}:{pi} interactions between enzyme and substrate at this position. We next addressed the significance of the residues at position 128/135/146 in the three deiodinases by creating the S128P D1, P135S D2, and P146S D3 proteins (Table I). Remarkably, replacement of the Pro-135 in D2 resulted in a two-order of magnitude increase in Km(T4) to ~250 nM, just ~10-fold lower than that of D1 for T4 (Table I). Double-reciprocal plots of product formation with T4 as the variable substrate at several dithiothreitol concentrations (0.5-4 mM) yielded patterns of parallel lines, indicating ping-pong kinetics (Fig. 5A). Furthermore, the D2-catalyzed deiodination became PTU-sensitive (Ki = 4.0 µM), although PTU inhibition was noncompetitive with dithiothreitol (Fig. 5B). Thus, the substitution of Ser for Pro-135 in D2 results in changes in the enzyme that make its kinetics more similar to those of D1, indicating a critical influence of the amino acid occupying this position on enzyme function. Similar observations were made with the P146S D3 protein (Table I), which displays a 5-fold higher Km(T3) and is highly sensitive to inhibition by PTU (Ki = 1.0 µM).


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  		TABLE I
Kinetic properties of wild type and mutated D1, D2 and D3 enzymes

A * denotes a construct prepared and characterized in the present investigation. Human templates were used for the mutation analyses. Km (rT3), Km (T4) or Km (T3) was used as a crude estimate of the effects of specific point-mutations in affinity of the D1, D2, or D3 enzymes for their respective optima substrates. PTU insensitivity is <30% inhibition at 10 mM PTU. The mechanism of deiodination was determined by the double-reciprocal plot of product formation with rT3 (D1) or T4 (D2) as the variable substrates done at several fixed DTT concentrations. Whenever applicable, 35S-labeled enzymes were analyzed by SDS-PAGE to confirm expression of inactive en-zymes (12). Correlation of positions among D1, D2, and D3 is shown in Fig. 4.

 


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  		FIG. 5.
Kinetic analyses of mutated D1 and D2 proteins. Deiodinase activities were measured in sonicates of HEK-293 cells transiently expressing the indicated proteins. In A, the D2-catalyzed deiodination follows ping-pong kinetics, and in B, it is uncompetitively inhibited by PTU, whereas in C, the D1-catalyzed deiodination follows a sequential kinetics.

 

Although the Km (rT3) (~0.2 µM) (preferred over T4 as a D1 substrate) and the ping-pong kinetics are not altered in the S128P D1 protein (Fig. 5C), the modified enzyme-catalyzed deiodination became resistant to PTU (Ki > 1 mM), suggesting there was no longer an accessible E-Se-I intermediate, again illustrating the pivotal role of this position in the deiodinase molecule. The presence of Pro in the 128/135/146 position could result in tighter binding of the substrate in the D2 (and D3) binding pockets, perhaps explaining the ~1,000-fold higher substrate affinity of D2 and D3 than for D1. This could reflect an interaction between the 4'-OH group of the different iodothyronines and the Pro residue at the 128/135/146 position, which could explain why sulfate conjugation of this group dramatically increases the Vmax/Km and changes the site of T4 deiodination from an outer to an inner ring iodine (34).

The region of the deiodinase pocket defined by the {beta}2 motif of the TRX fold and the IDUA-like insertion was also explored (Fig. 4). Three conserved amino acids with charged polar side chains mark the transition between {beta}2 and the helix in the IDUA-like insertion, Glu/Asp-155, Glu-156, and His-158 (D1 residues; see Fig. 4B for deiodinase-specific numbering). We first targeted the invariant Glu-156 (D1), Glu-163 (D2), and Glu-174 (D3), replacing it with Ala. The resulting enzymes have no deiodinase activity (Table I). Replacement of Glu-156 with Asp in D1 supported deiodination but with a ~4.5-fold higher Km(rT3), whereas a similar substitution at position 163 in D2 does not affect Km(T4) (Table I). Thus, the acidic amino acids in this region of the deiodinase pocket are important for enzyme function and/or substrate binding, although the length of the side chain can vary. This is further supported by previous mutational studies of the invariant His position 158 in D1 (35). Its mutation to Asn, Gln, or Phe resulted in complete loss of deiodinase activity. Replacement of the corresponding His-165 in D2 with Asn also results in loss of deiodinase activity (Table I). According to the model, residues in this acidic pocket could interact with either NH3+/COOH-group in Ala side chain of the iodothyronines, an hypothesis supported by previous studies indicating that the positively charged T4 analog (3,5,3',5'-tetraiodothyroethylamine), which lacks a COOH-, is not a D1 substrate (36). In addition, the highest affinities for D1 (lowest apparent Km values) are those compounds in which positively charged functional groups (NH3+) are absent, such as tetraiodothyroacetic acid. This is further supported by the finding that the Km(T4) values for D- and L-T4 are not different (36). These data argue that the COO-group in the iodothyronines interacts with the NH3+ group of His in the 158 (D1) position and that the other acidic residues in this pocket act to reduce the ionization of the His residue. The critical role played by the IDUA-like insertion is further strengthened by the complete loss of deiodinase activity when the conserved Trp-163 (D1) and the corresponding Trp-170 in D2 are replaced with Ala (Table I).

Furthermore, amino acid substitutions in positions further away from the acidic pocket, such as the invariant H174N (D1) (Table I), results in an active D1 protein but with an ~20-fold increased Km(rT3) (35). A similar Gln substitution for His-185 in D2 does not alter its Km(T4) (Table I). Other residues in the TRX-fold IDUA-defined deiodinase active center are less critical for substrate binding. Results of these mutations are described in Table I and include, relative to D1, those in the {beta}1 motif (Phe-121, Cys-124) or in positions more COOH-terminal to the IDUA-like insertion such as in the {beta}3 motif (Glu-214).

Despite the above evidence identifying and characterizing the substrate binding pocket of the deiodinases as the structure formed by the {beta}1-{alpha}1-{beta}2 motifs of the TRX fold and the IDUA-like insertion, the lack of a structural model for the N-linker domain of the three deiodinase proteins (Fig. 1) does not allow an explanation for the previously demonstrated important role of the aromatic ring of Phe-65 in enhancing the binding of rT3 but not T4 (37). This could indicate that distant residues may also contribute importantly to substrate binding.

Significance of the Deiodinase/Glycoside Hydrolase Structural Similarity—The previously unrecognized similar topology between {beta}1-{alpha}1-{beta}2-{alpha}2-{beta}E of the GPX-like TRX fold and {beta}6-{alpha}6-{beta}7-{alpha}7-{beta}8 of the clan GH-A of glycoside hydrolases (Fig. 3) may only concern a pure structural relationship around a common {beta}-{alpha} motif with {beta}-strands being part of a parallel {beta}-sheet (open for the TRX fold and circularly closed for the clan GH-A TIM barrel). This puzzling observation may suggest that such enzyme structures made of {beta}-sheets of parallel {beta}-strands could have evolved from the use of a primitive supersecondary structure consisting in the succession of a {alpha}-helix and a {beta}-strand, the COOH-terminal end of which bears functional active residues. The evolution of ({alpha}/{beta})8 barrels by duplication and fusion of an ancestral half-barrel (discussed in Ref. 38) supports such a hypothesis. However, in this case, the striking sequence similarity of the deiodinase active site region with members of the clan GH-A, in particular within the IDUA-like IOD active-site insertion predicted to form a cap as in haloalkane dehalogenases, questions to what extent an evolutionary process of sequence concatenation may have occurred under the pressure of roughly similar substrates. This new example of unique evolutionary path could be further discussed in the light of the new protein evolution and folding brought by the recent discovery of "closed loops" (Ref. 39 and references therein).

It is perhaps not unexpected that the three selenodeiodinases are TRX-fold family proteins in light of the fact that GPX and other Sec-containing thiol-interacting oxidoreductases are part of this group (18). However, it is remarkable that selenodeiodinases have a IDUA-like sequence embedded within the Sec-containing TRX fold, which is critical for iodothyronine binding as it is that of iduronic acid sulfate in the GH-A family of proteins. Our results show that amino acid variations in the otherwise generally well conserved TRX-IDUA active center can explain important differences between D1 and D2. Although further refinements of this model are required, we believe that it provides an important first step in the structural analysis of the rate-limiting enzymes controlling thyroid hormone activation.


   FOOTNOTES
 
* This work was supported by DK 36256 and DK58538 grants from the NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

A Magyary Zoltán Postdoctoral Fellow of the Hungarian Education Ministry and supported by a Felsõoktatási Kutatási és Fejlesztési Pályázat (FKFP 0036/2001) grant and the Fifth EC Framework Program (QLG3 2000-00844). Back

{ddagger}{ddagger} To whom correspondence should be addressed: Brigham and Women's Hospital, 77 Avenue Louis Pasteur, HIM Bldg. 643, Boston, MA 02115. Tel.: 617-525-5153; Fax: 617-731-4718; E-mail: abianco{at}partners.org.

1 The abbreviations used are: HCA, hydrophobic cluster analysis; TRX, thioredoxin; GH, glycoside hydrolase; Sec, selenocysteine; TM, transmembrane; HEK, human embryonic kidney; wt, wild type; GPX, glutathione peroxidase; IDUA, iduronidase; PTU, 6n-propyl-2-thiouracil. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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