Crystal structure of mammalian selenocysteine-dependent iodothyronine deiodinase suggests a peroxiredoxin-like catalytic mechanism

Abstract

Local levels of active thyroid hormone (3,3',5-triiodothyronine) are controlled by the action of activating and inactivating iodothyronine deiodinase enzymes. Deiodinases are selenocysteine-dependent membrane proteins catalyzing the reductive elimination of iodide from iodothyronines through a poorly understood mechanism. We solved the crystal structure of the catalytic domain of mouse deiodinase 3 (Dio3), which reveals a close structural similarity to atypical 2-Cys peroxiredoxin(s) (Prx). The structure suggests a route for proton transfer to the substrate during deiodination and a Prx-related mechanism for subsequent recycling of the transiently oxidized enzyme. The proposed mechanism is supported by biochemical experiments and is consistent with the effects of mutations of conserved amino acids on Dio3 activity. Thioredoxin and glutaredoxin reduce the oxidized Dio3 at physiological concentrations, and dimerization appears to activate the enzyme by displacing an autoinhibitory loop from the iodothyronine binding site. Deiodinases apparently evolved from the ubiquitous Prx scaffold, and their structure and catalytic mechanism reconcile a plethora of partly conflicting data reported for these enzymes.

Keywords: iodothyronine deiodination; selenenyl-sulfide; selenoprotein; thiol cofactor; thioredoxin fold.

Fig. 1.

Crystal structure of Dio3^cat. (A) Chemical structure of T₄ with numbering of iodine positions (underlined). (B) Overall structure of the Dio3 catalytic domain, with secondary structure elements present in Trx in cyan and labeled according to convention. The N-terminal Prx-like module is highlighted in magenta, the Dio-insertion is highlighted in blue, and α2 is highlighted in green. (C) Dio3^cat topology diagram. Catalytically relevant amino acids are labeled. (D) Active site region of Dio3^cat, with residues conserved and/or involved in catalysis shown in stick presentation. Sec^170* indicates the catalytic selenocysteine replaced by cysteine in our construct. (E) Close-up view of a model for Dio3 in complex with iodothyronine ligand, based on the Arg-T₃-His clamp in T₃Rβ (Dio3 substrate would be T₄). (F) 5-Deiodinase activity of WT Dio3 and mutants with changes in the α2/β3- and β4/α3-loops in the presence of DTT. Error bars indicate SEM (n = 3).

Fig. 2.

Dimerization of Dio3. (A) Homodimerization of full-length Dio3. C-terminally epitope-tagged deiodinase proteins (calculated masses of ∼37 kDa) were transiently expressed in HEK cells and analyzed by Western blotting after reducing SDS/PAGE. Immunoprecipitation (IP) of Flag-tagged Dio3 yields coprecipitated V5-tagged Dio3. Inclusion of T₄ does not enhance dimerization. IB, immunoblot. (B) Blue-native PAGE of recombinant His-tagged Dio3^cat (calculated mass of 26 kDa) and of monomeric size standards. Dio3^cat migrates in two bands, corresponding to the size of a monomer and dimer, respectively. (C) Close-up view of a modeled Dio3^cat iodothyronine complex, showing the clash with the α2/β3-loop and the shielding of Sec^170* by Phe²⁵⁸. (D) Surface of Dio3^cat, colored according to residue conservation in an alignment of Dio1–Dio3 enzymes from five different mammals. Magenta indicates high sequence conservation, and cyan indicates high variation. (E) Model for a full-length Dio3 dimer. A catalytic domain dimer was based on HBP23 and fused to a homology model for the transmembrane region. C-term, C terminus; N-term, N terminus.

Fig. 3.

Dio3 structure suggests a catalytic hydrogen bond network and a Prx-like reduction cycle. (A) Model for a Dio3 complex with iodothyronine (Fig. 1E). The residues of the conserved hydrogen-bond network putatively conveying a proton or preparing a water molecule for protonation at the deiodination site are labeled. Arrows indicate electron shifts for a potential proton relay. (B) T₄ 5-deiodination activity of Dio3 WT, Ser167Ala, Thr169Ala, Thr169Ser, Tyr197Phe, and Glu200Thr in the presence of 5 mM DTT. Sec170Ala served as a negative control. Mean ± SEM. (C) Overlay of Dio3^cat (magenta) with reduced PtGPX5 (green). Peroxidatic residue and resolving cysteines are indicated. (D) Comparison of the oxidized PtGPX5 form (green) with its reduced state (Left) and comparison of the model of Dio3^cat with selenenyl-sulfide (magenta) with the structure of the reduced enzyme (gray, Right). (E) Dio3 activity is supported by physiological thiol regenerating systems based on Grx-1 or Trx-1. (F) Dio3 T₄ 5-deiodination activity in presence of a Trx or Grx regenerating system, expressed as a percentage of WT Dio3 activity. Error bars indicate SEM (n = 3). 100%, 37–41 fmol of rT₃ from T₄ per milligram of protein per minute. (WT Dio3 with DTT: 855 fmol/(min * mg). (G) Reactivity of Dio3-Sec¹⁷⁰ in the absence of added thiols. Selenocysteine was derivatized with biotinylated iodoacetamide (BIAM) after addition of T₄. The BIAM signal was normalized to the Flag-Dio3 signal and is expressed as a percentage of WT Dio3. Error bars indicate SEM (n = 5). (H) Interaction of TrxCys35Ser with Dio3. Trx was coimmunoprecipitated with Flag-Dio3 WT or mutant and resolved in reducing SDS/PAGE, and the intensity for Trx was normalized to the respective Flag signal. A.U., arbitrary unit.