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Home , Iodothyronine deiodinase, Selenocysteine

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

Ulrich Schweizera,1,2, Christine Schlickerb,1, Doreen Brauna, Josef Köhrlec, and Clemens Steegbornd,2

aInstitut für Biochemie und Molekularbiologie, Rheinische Friedrich Wilhelms-Universität Bonn, 53115 Bonn, Germany; bDepartment of Physiological Chemistry, Ruhr University Bochum, 44801 Bochum, Germany; cInstitut für Experimentelle Endokrinologie, Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany; and dDepartment of Biochemistry and Research Center for Bio-Macromolecules, University of Bayreuth, 95440 Bayreuth, Germany

Edited* by Dieter Söll, Yale University, New Haven, CT, and approved June 9, 2014 (received for review December 23, 2013)

Local levels of active (3,3′,5-) are of Mus musculus Dio3 (Dio3cat)withSec170 replaced by controlled by the action of activating and inactivating iodothyro- (Fig. 1 B and C and Table 1). The structure was solved by nine deiodinase . are selenocysteine-depen- single-wavelength anomalous dispersion phasing using crystals of cat dent membrane catalyzing the reductive elimination of (SeMet)-labeled Dio3 . Refinement of native cat iodide from iodothyronines through a poorly understood mecha- and SeMet-labeled Dio3 resulted in structures without recog- nism. We solved the crystal structure of the catalytic domain of nizable differences, neither for the backbone (rmsd for 186 Cα mouse deiodinase 3 (Dio3), which reveals a close structural simi- positions = 0.2 Å) nor for side chains, with slightly better statistics larity to atypical 2-Cys peroxiredoxin(s) (Prx). The structure suggests for the SeMet structure (Table 1). Further analyses were thus done a route for proton transfer to the substrate during deiodination and with the SeMet structure refined at a resolution of 1.9 Å to R/Rfree a Prx-related mechanism for subsequent recycling of the transiently values of 18.7% and 22.7%, respectively. oxidized . The proposed mechanism is supported by bio- cat chemical experiments and is consistent with the effects of muta- Overall Deiodinase Structure and Substrate . Dio3 tions of conserved amino acids on Dio3 activity. Thioredoxin and adopts a thioredoxin (Trx) fold (6) as previously predicted (7), reduce the oxidized Dio3 at physiological concentra- with deiodinase-specific modifications and insertions, resulting tions, and dimerization appears to activate the enzyme by displac- in architecture with a five-stranded, mixed β-sheet flanked by ing an autoinhibitory loop from the iodothyronine binding site. four α-helices (Fig. 1 B and C). N-terminal to the Trx βαβ-motif Deiodinases apparently evolved from the ubiquitous Prx scaffold, lies a small, two-stranded, antiparallel β-sheet, βN, followed by Θ and their structure and catalytic mechanism reconcile a plethora of a short 310-helix, 1. The essential deiodinase-specific insertion partly conflicting data reported for these enzymes. (Dio-insertion, residues 201–225; Fig. 1 B and C) forms a large loop-D, followed by a helix αD and a short βD that aligns with β iodothyronine deiodination | thioredoxin fold | | the central mixed -sheet. The loop, which is critical for iodo- thiol | selenenyl-sulfide thyronine binding and was previously called an iduronidase-like insertion, forms a compact protrusion instead of the predicted hyroid regulate mammalian development as well Tas energy expenditure and metabolism in the adult (1). The Significance active, nuclear -binding form of thyroid hormone is Deiodinases activate and inactivate through 3,3′,5-triiodothyronine (T3). The thyroid gland mainly releases a unique biochemical reaction. Enzymes expand their catalytic thyroxine [3,3′,5,5′-tetraiodothyronine (T4); Fig. 1A], and T3 is formed and degraded through elimination of atoms from capabilities through special heteroatoms in cofactors or in the the 5′- and 5-positions, respectively (2) (Fig. S1). Three types of rare but essential selenocysteine, and deiodinases deiodinase enzymes are involved in activation and inactivation use an active-site selenocysteine for the reductive elimination of thyroid hormones (Fig. S1). They form a family of trans- of iodide from the aromatic iodothyronine rings. The mecha- membrane enzymes with homologous catalytic domains (2). nism of deiodinases has remained elusive despite many mu- Type II deiodinase (Dio2) catalyzes the activating (outer ring) tational and enzymatic studies. We solved the crystal structure of the deiodinase catalytic domain and find that it resembles 5′-deiodination of the prohormone T4 to T3, whereas type III deiodinase (Dio3) catalyzes inactivating (inner ring) 5-deiodination a family of peroxiredoxin(s) (Prx). Structure and biochemical (2). Type I deiodinase (Dio1) is capable of both types of reac- data suggest a deiodinase catalytic mechanism with Prx-like tions (3). Cells targeted by the hormone can thereby fine-tune elements and enable us to assign unexpected functions to residues previously reported to contribute to deiodinase ca- intracellular T3 levels through deiodinase expression according to their needs during development or upon metabolic challenges. talysis. Our findings indicate how deiodinases may have evolved Dio1, Dio2, and Dio3 share a conserved amino acid sequence from a common reductase ancestor. and a selenocysteine residue essential for efficient deiodination, Author contributions: U.S. and C. Steegborn designed research; C. Schlicker and D.B. although nonmammalian cysteine-deiodinases have been found performed research; U.S., C. Schlicker, D.B., J.K., and C. Steegborn analyzed data; and (4, 5). Although most of the characterized act U.S., C. Schlicker, D.B., J.K., and C. Steegborn wrote the paper. as or reductases, deiodinases are the only The authors declare no conflict of interest. selenoenzymes known to catalyze halogen eliminations from *This Direct Submission article had a prearranged editor. aromatic rings. Despite a large body of experimental data, there Data deposition: The atomic coordinates and diffraction data have been deposited in the is no coherent model for deiodinase catalysis that incorporates Protein Data Bank, www.pdb.org (PDB ID codes 4TR3 and 4TR4). the bulk of available data. 1U.S. and C. Schlicker contributed equally to this work. 2To whom correspondence may be addressed. Email: [email protected] or clemens. Results and Discussion [email protected]. To explore the unique deiodinase catalytic mechanism, we de- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. termined the crystal structure of the cytoplasmic catalytic domain 1073/pnas.1323873111/-/DCSupplemental.

10526–10531 | PNAS | July 22, 2014 | vol. 111 | no. 29 www.pnas.org/cgi/doi/10.1073/pnas.1323873111 Downloaded by guest on September 30, 2021 Fig. 1. Crystal structure of Dio3cat.(A) Chemical structure

of T4 with numbering of iodine positions (underlined). (B) Overall structure of the Dio3 catalytic domain, with sec- ondary structure elements present in Trx in cyan and la- beled 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) Dio3cat topology diagram. Catalytically relevant amino acids are labeled. (D) region of Dio3cat, with residues conserved and/or involved in catalysis shown in stick presentation. Sec170* indicates the catalytic seleno- cysteine replaced by cysteine in our construct. (E) Close-up view of a model for Dio3 in complex with iodothyronine

, based on the Arg-T3-His clamp in T3Rβ (Dio3 sub- strate 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).

α-helix (7). The catalytic Sec170 (replaced by a cysteine in our Dio3-His202, which corresponds to the catalytically essential Dio1- construct, as indicated by *) is positioned in the loop connecting His158, protrudes from the Dio-insertion into the substrate binding β1toα1, in the same position as the peroxidatic cysteine of thiol cleft (Fig. 1D). Its distance to the catalytic selenocysteine and reductases with a Trx-like fold [Trx, (Prx), and position at the end of this cleft suggest that rather than acting glutathione (GSH) peroxidases GPX (see below); Fig. 1D]. It catalytically, His202 serves as a binding partner for the 4′-phe- 435 points toward an elongated cleft that likely represents the iodo- nolic end of the substrate, similar to His in the T3-receptor β 435 282 thyronine binding site. Two conserved deiodinase are (T3Rβ) complex (9). Superimposing the His -T3-Arg clamp 158 174 202 275 important for Dio1 activity, His and His (8). The side chain from the T3/T3Rβ complex on Dio3-His -Arg provides a first of Dio3-His219 (corresponding to Dio1-His174) is buried between substrate complex model and places the iodothyronine into the loop-D and helix αD and appears to contribute to catalysis proposed Dio3 substrate binding cleft (Fig. 1E). The targeted 5- through an interaction with the conserved Glu200 (see below). iodine atom is positioned ∼3–4 Å from the Se atom of Sec170,

Table 1. Data collection and refinement statistics SeMet Dio3 Native Dio3

Space group P212121 P212121 Unit cell constants a = 48.9 Å, b = 54.3 Å, a = 48.8 Å, b = 54.2 Å, c = 66.6 Å; α, β, γ = 90° c = 66.5 Å; α, β, γ = 90° Resolution 42.07–1.90 Å 42.01–1.93 Å Unique reflections 14,304* 13,799 14.9 (5.3) 11.9 (3.6) † Completeness 95.5% (72.6%) 98.5% (89.5%) †,‡ Rmerge 7.3% (24.1%) 8.6% (34.8%) Se sites 4 —

CC Eobs to Ecalc(Se) 35.7% — Pseudofree CC 64.6% — Refinement resolution 1.90 Å 1.93 Å Reflections used for refinement 13,309 12,919 Protein atoms 1,462 1,461 Solvent atoms 93 114 rmsd bond lengths 0.015 0.015 rmsd bond angles 1.4 1.5 Average B-factor 18.0 Å2 16.4 Å2 §,{ Final R/Rfree 18.7%/22.7% 19.1%/23.4% BIOCHEMISTRY

CC, correlation coefficient; Ecalc, calculated normalized structure factors; Eobs, observed normalized structure factors; , intensity normalized by standard deviation. *Value for merged Friedel pairs. Before merging: 25,930. † NumbersP in parentheses are for the outermost shell. ‡ PðI − hIiÞ Rmerge = ; I is the intensity of an individual measurement, and hIi is the corresponding mean value. P I jj j − j jj § FPobs k Fcalc R-factor = ; jFobsj is the observed structure factor amplitude, and jFcalcj is the calculated struc- jFobs j ture factor amplitude. { Rfree was calculated from 5% of measured reflections omitted from refinement.

Schweizer et al. PNAS | July 22, 2014 | vol. 111 | no. 29 | 10527 Downloaded by guest on September 30, 2021 compatible with molecular orbital calculations for 5-deiodination 2E] results in close contacts between the N-terminal loop and β4of through selenenyl-iodide formation (see below) (10). The car- one monomer and the β4/α3-loop and substrate site of the second boxyl group of the substrate is positioned close to the guanidi- monomer, which may mediate relaxation of the α2/β3-loop, and nium group of Dio3-Arg275 situated in the β4/α3-connecting loop thus allow substrate binding (Fig. 2 C and E). In contrast, the 196–198 (Fig. 1E). In line with this T3R-like substrate pinching by a his- conserved Ile-Tyr-Ile motif previously proposed to mediate tidine- clamp (9), which was also proposed for the T3 dimerization (12) is hidden in the enzyme’s hydrophobic core and transporter monocarboxylate transporter 8 (11), 5-deiodinase appears to contribute to Dio3cat stability and activity (see below). activity is sharply reduced in a Dio3-Arg275Ala mutant (Fig. 1F). Moreover, Dio3-Glu259 would be properly positioned for rec- Proton Transfer During Reductive 5-Deiodination. Our Dio3 struc- ognizing the iodothyronine amino group. Mutating the corre- ture supports a previously proposed deiodinase half-reaction that 214 sponding Glu in Dio1 increased the substrate Km, supporting leads to the formation of a reduced deiodinated substrate and such a role in iodothyronine binding (7). concomitant formation of an oxidized selenenyl-iodide enzyme intermediate (16). This deiodination step is supported by density Deiodinase Dimerization. Deiodinase activity is reported to require functional theory studies suggesting a selenolate inline attack on dimerization partly mediated by the catalytic domain but criti- the iodine σ-hole weakening the carbon–iodine bond. The ab- cally depending on the N-terminal transmembrane anchor and stracted iodonium is replaced by a proton approaching from the linker region (12). Indeed, full-length Dio3 formed dimers (Fig. opposite side of the ring (10). Our structure suggests that the 2A), whereas our N-terminally truncated recombinant Dio3cat proton is conveyed to the 5-position of the iodothyronine along protein behaved as a monomer in gel filtration and mainly mo- atriadHis219,Glu200, and Ser167 (Fig. 1E), with residues Tyr197 and nomeric, with little dimeric species, in native gel electrophoresis Thr169 participating in the organization of an intricate H-bond (Fig. 2B). The catalytic domain itself thus has weak dimerization network (Fig. 3A). This mechanism explains the conservation of capabilities but requires support by the N-terminal region to Glu200 and His219, as well as previous mutagenesis results on these form stable dimers. All protein/protein interactions observed in two residues in Dio1 and Dio2 (7, 8). In addition, it explains how the Dio3cat crystal structure appear to represent crystal contacts, Glu200 and His219 can contribute to catalysis despite their distance and the structure thus constitutes an inactive monomeric form. to the reacting iodine. To probe the catalytic relevance of Glu200 In this inactive state, Phe258 from the α2/β3-loop protrudes into and the other network residues, we tested the activities of several the substrate-binding cleft around Sec170 and blocks the active Dio3 mutants (Fig. 3B). Removing the acidic function in Dio3- site (Fig. 2C). Consistently, soaking Dio3cat crystals with iodo- Glu200Thr inactivated the enzyme completely. The same result thyronine substrate or with the inhibitor did not was obtained for removing the interacting hydroxyl groups in result in electron density for a ligand. Phe258 is the only Dio3cat Tyr197Phe and in Thr169Ala (Fig. 3 A and B). A Thr169Ser mu- residue in the disallowed region of the Ramachandran plot, yet tant, in contrast, showed only ∼60% reduced activity compared it is well defined by electron density. The α2/β3-loop, known to with WT Dio3, consistent with the catalytic relevance of the resi- contribute to substrate binding in other Trx-fold proteins (13), due’s hydroxyl group and with the best suitability of a in thus appears to assume a strained, autoinhibited conformation. this position indicated by the absolute conservation of Thr169 in We speculate that the loop is relaxed upon dimerization, deiodinases. Mutation of Ser167, which is also conserved among allowing access to the binding site and the reactive Sec170 that deiodinases and even in Prx (see below) to Ala decreased activity by is, until then, shielded by Phe258. When we limited its con- >90% (Fig. 3B). Ser167 is positioned next to the substrate pro- formational flexibility by a Phe258Pro mutation, 5-deiodinase tonation site and forms the end point of the catalytic H-bond activity was indeed repressed (Fig. 1F). Likewise, mutation of network (Figs. 1D and 3A). Although the exact catalytic function of Tyr257 to Ala inactivated the enzyme, indicating that its ac- this network remains to be clarified, our analyses suggest that it tivity is sensitive to changes of the α2/β3-loop sequence (Fig. conveys a proton or prepares a water molecule for protonation 1F). Interestingly, several amino acids conserved across dei- of the iodothyronine upon deiodination (Fig. 3A). odinases and exposed to the surface, but not directly involved in substrate binding or catalysis, cluster around β4 and α3 (Fig. Structural Similarity to Prx Proteins. Due to its instability, a sele- 2D). This region mediates dimerization in the redoxins heme- nenyl-iodide intermediate has not been observed in deiodinases binding protein 23 kDa (HBP23) and Populus trichocarpa X del- and has only recently been detected in a sterically protected toids GPX-like (PtGPX5), structural homologs of model compound (17). Whether the selenenyl-iodide may hy- Dio3cat (see below) (14, 15). Modeling a Dio3cat dimer based on an drolyze in an aqueous environment to form a selenenic acid, in HBP23 template [Protein Data Bank (PDB) ID code 1QQ2; Fig. analogy to the sulfenic acid intermediate in Prx (18), remains to

Fig. 2. Dimerization of Dio3. (A) Homodimeriza- tion 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, immuno- blot. (B) Blue-native PAGE of recombinant His- tagged Dio3cat (calculated mass of 26 kDa) and of monomeric size standards. Dio3cat migrates in two bands, corresponding to the size of a monomer and dimer, respectively. (C) Close-up view of a modeled Dio3cat iodothyronine complex, showing the clash with the α2/β3-loop and the shielding of Sec170* by Phe258.(D) Surface of Dio3cat, 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.

10528 | www.pnas.org/cgi/doi/10.1073/pnas.1323873111 Schweizer et al. Downloaded by guest on September 30, 2021 be clarified. In fact, there is no consensus on the mechanism for the first turn of α1 and the destabilized αD of the reduced state the entire second deiodinase half-reaction (i.e., iodide release, locally unfold and enable a disulfide bridge between Cys44 and reduction of the oxidized enzyme). The deiodinase crystal Cys92 (15). In the reduced state, the resolving cysteine PtGPX5- structure now suggests an answer to this question. The Trx su- Cys92 and Dio3-Cys239 from the respective αD/βD-loop region perfamily comprises protein families with different functions, show -sulfur and sulfur-selenium distances to their perox- characterized by family-specific modifications to the Trx fold (6). idatic residue of 21 Å (PtGPX5) and 17 Å (Dio3), respectively. Comparison of Dio3cat with known structures reveals close Some missing side chains and high B factors in the Dio3cat Dio- structural similarity to proteins related to atypical 2-Cys Prx, such insertion suggest conformational flexibility, consistent with a Prx- as the poplar thiol peroxidase (Tpx) PtGPX5 (15) and the like rearrangement of loop-D and the αD C terminus to allow Escherichia coli Tpx (19) (Fig. 3 C and D). A comparison of formation of a Cys239-Sec170 selenenyl-sulfide. In fact, modeling an Dio3cat and PtGPX5 (PDB ID code 2P5Q) with Prx shows that oxidized Dio3cat state required only very moderate rearrange- Dio3 and PtGPX5 differ only in a C-terminally longer α2 and ments of these regions (Fig. 3D). We thus propose that Cys239 may a shorter loop-D in PtGPX5 (Fig. 3C) and that they indeed serve as a resolving cysteine in a Prx-like deiodination mechanism feature the two characteristics of the Prx family. An additional with a selenenyl-sulfide as a possible intermediate state. helix in the variable Trx-fold insertion, αD in the Dio-insertion, is present in Prx but also in some other Trx-fold protein families Prx-Like Deiodinase Reduction Half-Cycle. Oxidized 2-Cys Prx, (6). The N-terminal βN1/2-Θ1 module found in Dio3cat (Fig. Tpx, and PtGPX5 are reduced by Trx. In vitro deiodinase as- 1C), however, is highly characteristic for Prx. The evolutionary says usually use the nonphysiological reducing agent DTT, but and mechanistic relationship between deiodinase and Prx sug- Goswami and Rosenberg (21) purified from cells a protein thiol gested by these structural characteristics is further supported by cofactor resembling glutaredoxin (Grx). Another group identi- a Ser-X-X-Sec motif conserved in all deiodinases. It corresponds fied Trx and Trx reductase (TrxR) as cytosolic factors needed to a Ser/Thr-X-X-Cys motif known as a hallmark of all Prx, which for Dio1 reduction (22–24), consistent with decreased Dio1 ac- comprises the peroxidatic cysteine and a catalytically important tivity under conditions decreasing reduced Trx (25). Dio1 activity Ser/Thr (18, 20). We find that the corresponding Ser167 in Dio3 from measured with a physiological NADPH-dependent is indeed essential for deiodinase activity (Fig. 3B). In atypical 2- Trx/TrxR regenerating system was lower than with millimolar Cys Prx, a reduced product and an intramolecular disulfide be- DTT but revealed a Km(Trx) of 1.8 μMandaKm[reverse T3 (rT3)] tween the peroxidatic cysteine and a resolving cysteine can be of 1.4 nM, as well as sequential kinetics as in Dio2 and Dio3 (24). formed during the first catalytic half-reaction, and the disulfide is Here, we tested whether physiological regenerating systems subsequently reduced by Trx. Dio3-Sec170* superimposes with containing 3 μM Trx-1 and 0.1 μMTrxreceptor1or1μMGrx-1, the peroxidatic Cys44 in reduced PtGPX5, and Dio3-Cys239, which 1 mM GSH, and 0.1 μM GSH reductase are capable of sustaining is conserved in all deiodinases, is located in the αD/βD-loop Dio3 activity. Both systems supported Dio3 activity in an 92 similar to the conserved resolving Cys of PtGPX5 (Fig. 3C). In NADPH-dependent manner (Fig. 3E), and the Km(T4)is6.7nM thecrystalstructureofoxidizedPtGPX5(PDBIDcode2P5R), inthepresenceofTrx.GSHaloneisabletosustainDio3

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 Dio3cat (magenta) with reduced PtGPX5 (green). Peroxidatic residue and resolving are indicated. (D) Comparison of the oxidized PtGPX5 form (green) with its reduced state (Left) and comparison of the model of Dio3cat 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)Dio3T4 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 BIOCHEMISTRY 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 coimmu- noprecipitated with Flag-Dio3 WT or mutant and resolved in reducing SDS/PAGE, and the intensity for Trx was nor- malized to the respective Flag signal. A.U., arbitrary unit.

Schweizer et al. PNAS | July 22, 2014 | vol. 111 | no. 29 | 10529 Downloaded by guest on September 30, 2021 activity but is required in concentrations above 10 mM for treatment with T4 substrate in the absence of reducing agent, significant activity, whereas1mMGSHissufficientinthe indicating that the selenenic acid resulting from deiodination presence of Grx-1 and the regenerating system (Fig. 3E). We canbereducedbeforeinteractionwithanexternalreductant. thus conclude that analogous to Prx, deiodinases can be reduced Mutating Cys239 and/or Cys168 decreased the labeling by the physiological protein thiols Trx-1 and/or Grx-1 coupled to (Fig. 3G), suggesting an intrinsic selenol regeneration mecha- their cognate regenerating systems. nism involving both conserved cysteines, the proximal Cys168 as We next analyzed the two cysteines conserved in deiodinases. well as Cys239, which is positioned remote from the active site The putative resolving Cys239 is conserved even in distantly re- in the reduced enzyme (Fig. 3D). The requirement of both cys- lated nonvertebrate deiodinases (5), whether containing an ac- teines suggests a selenenic acid reduction cascade with a 170–239 tive-site selenocysteine or cysteine (Fig. S2). When DTT was selenenyl-sulfide isomerizing to a 168–239 disulfide, which might 194 used as a reductant, mutation of the Dio1 resolving Cys re- be more accessible for the reducing thiol. Preferential interaction V sulted in decreased max, which was further decreased when of Trx with a 168–239 disulfide is supported by Dio3 cross-links GSH or Trx was used (26). We generated several Dio3 mutants with a Trx-Cys35Ser variant. WT deiodinase was efficiently cross- and tested their activities with our Trx- or Grx-dependent re- linked, but mutation of either Cys239 or Cys168 reduced this in- generating systems (Fig. 3F). The activity of Dio3-Cys239Ala teraction (Fig. 3H), suggesting that both cysteines contribute to appeared unchanged compared with WT. That the peroxidatic covalent Trx binding. Overlaying Dio3cat with a human Trx/Trx- residue of a Trx-dependent peroxidase can be directly reduced reductase complex (PDB ID code 3QFB) (32) indicates Trx binding in the presence of a sufficient thiol partner when the resolv- around the αD C terminus (Fig. S3), and a 168–239 disulfide ap- ing cysteine is mutated is not without precedence (18, 27). A pears slightly more accessible from this side (Fig. S6). The sug- Aeropyrum pernix Trx-dependent Prx from can be reduced even if gested deiodinase mechanism resembles disulfide cascades in Prx the resolving cysteine is removed (28). For this enzyme, two reduced carrying three conserved cysteines (28, 33) and is consistent with states were observed, one of them resembling the conformation the observation that mutating the proximal Dio3 cysteine in- of its disulfide-bridged, oxidized form (PDB ID code 2CX4), so creases the Km for the reducing thiol (26, 30) and/or reduces the that reduction of the oxidized peroxidatic residue may take place overall velocity of the reaction (Fig. 3F). in the oxidized conformation without prior disulfide formation. Previous results support significant conformational changes Similarly, Trypanosoma brucei PrxII was shown to sample the 128 “ ” during catalysis: Mutation of Dio1-Ser (equivalent position to disulfide conformation already in its reduced state and to serve Dio3-Pro172; Figs. S2 and S3) to changes the observed ki- as a substrate for reduction without resolving cysteine and netics of Dio1 from ping-pong to sequential (7). Vice versa, mu- disulfide formation (18, 29). A cross-link of peroxidatic residue tation of Pro135 to Ser changes Dio2 kinetics to ping-pong (7). and resolving cysteines is thus proposed to protect the perox- These findings suggest that this residue modulates the kinetics of idatic group against overoxidation during the slow reduction local rearrangements at the α1 N terminus required for selenenyl- cycle rather than to trap the oxidized conformation for reduction 170 239 sulfide formation. Resolution of the selenenyl-sulfide by a proximal (18). An oxidized conformation in Dio3 with a Sec -Cys cysteine only in Dio3 and Dio1 would also explain the “molecular selenenyl-sulfide is consistent with selenol-labeling experiments memory” in Dio2, which marks the enzyme for ubiquitination after (see below) and with the so far enigmatic results with the Dio1- substrate deiodination (34, 35). The Dio2-specific insertion of 18 specific inhibitor propyl-thiouridine (PTU). PTU reacts with amino acids between βN1 and βN2 (destruction sequence) would Dio1 only after reaction with iodothyronine substrate and com- petes with the reducing thiol substrate. In the model of oxidized be in close contact to the part of the enzyme rearranged in the Dio3 (Fig. 3D and Fig. S3), Pro172 would shield the selenenyl- oxidized (selenenyl-sulfide) conformation (Fig. S7). GSH at con- sulfide from the bulky PTU. Consistently, mutating Pro172 to the centrationsupto25mMisunabletoreactivateoxidizedDio2(36). found in Dio1 (Ser128) renders Dio3 PTU-sensitive (7). In contrast, the nonphysiological dithiol DTT apparently can re- duce the selenenyl-sulfide, releasing the enzyme for repeated rounds of catalysis in vitro. In fact, mutating Dio2-Ala131 to cys- Potential Deiodinase Isomerization Cascade. In Dio1 and Dio3, K a second cysteine is conserved, proximal to the catalytic sele- teine reduced its m for DTT, supporting the conclusion that nocysteine within a SerCysThrSec motif. Mutating this Cys124 in a cysteine in this position facilitates the interaction with reducing V K thiols (36). In vivo, WT Dio2, without the proximal cysteine, may Dio1 reduced the max and increased the m for DTT 14-fold – (26, 30), leading to the conclusion that the proximal cysteine remain trapped in the 170 239-like selenenyl-sulfide conformation, interacts with the electron donor. Mutation of Dio3-Cys168 re- and is thus marked for degradation. duced 5-deiodinase activity by 50% in the presence of the Trx/ cat Grx regenerating systems (Fig. 3F), and kinetics in presence of Conclusions. The crystal structure of Dio3 confirms its Trx GSH indicated a reduction of the V (Fig. S4). Deiodination scaffold and suggests a dimerization interface involved in acti- max C studies with a model compound containing a selenolate and vation through release of an autoinhibitory loop (Fig. 2 and a nearby thiol suggested that a proximal thiol may specifically Fig. S6). Thereby, substrate binding to an arginine/ enhance 5-deiodination (31), which would be consistent with the clamp next to the catalytic selenocysteine is enabled. The conservation of this cysteine only in the deiodinases exhibiting structure further reveals deiodinase-specific features classifying inner-ring deiodination, Dio1 and Dio3 (Fig. S2). We find, them as evolutionarily related to atypical 2-Cys Prx. Mutation of the conserved Ser167 within Dio3’s Prx signature motif supports however, that a human DIO1-Cys124Ala mutant displays re- 170 duced 5′-deiodination activity against rT3 (Fig. S5) as previously a Prx-like catalytic mechanism. In this mechanism, Sec extracts reported (30), excluding a function specifically in inner-ring the 5-iodine as a selenenyl-iodide (16), which might hydrolyze deiodination. Instead, we suggest that the proximal cysteine is quickly, and the thyronine ring is protonated via a network of involved in reduction of Dio3 after initial formation of a Sec170- conserved amino acids (Fig. 3A and Fig. S6). The oxidized enzyme Cys239 selenenyl-sulfide. Although protected from overoxidation can be directly reduced by exogenous thiols in vitro. Structural (18), this state is likely not optimal for reduction. Cys168 points similarity of Dio3cat to atypical 2-Cys Prx suggests that physio- away from the active site in our structure of reduced Dio3cat, but logically, Dio3-Cys239 acts as a resolving cysteine and can form it appears properly oriented to attack a Sec170-Cys239 bond in this a selenenyl-sulfide with Sec170. Orientation of Cys168, the addi- selenenyl-sulfide form of the enzyme (Fig. 3D). A reaction of tional conserved cysteine in Dio1 and Dio3, and biochemical Cys168 with the selenenyl-sulfide is supported by experiments data suggest that it can further resolve the selenenyl-sulfide to a selectively labeling reduced with the alkylating agent Cys168-Cys239 disulfide that is more exposed and reduced more easily biotin iodoacetamide at low pH (Fig. 3G). Selectivity for Sec170 by protein thiols. The experimental deiodinase structure thus was confirmed by the complete loss of an alkylation signal for the explains many hitherto enigmatic features of deiodinase bio- Dio3-Sec170Ala mutant. WT Dio3 was efficiently alkylated after chemistry. The close relationship to Prx-like proteins further

10530 | www.pnas.org/cgi/doi/10.1073/pnas.1323873111 Schweizer et al. Downloaded by guest on September 30, 2021 suggests an evolutionary pathway explaining how the activity of REFMAC (42). The structures were analyzed with Procheck (43). Deiodinase has arisen, with a Prx as an ancestor. homology models were generated using Modeler (44). Oxidized Dio3cat was modeled with oxidized PtGPx5 (PDB ID code 2P5Q) as a template. The Dio3 Materials and Methods dimer was generated superimposing Dio3 on a Prx dimer (PDB ID code 1QQ2). Detailed experimental information is provided in SI Materials and Methods. For the full-length dimer model, the N-terminal transmembrane and linker part was generated with Phyre2 (45). To model a Dio3/Trx complex, Dio3 was Structure Solution and Homology Modeling. The active-site selenocysteine of positioned on top of Trx reductase of a Trx/Trx reductase complex (PDB ID code cat mouse Dio3 was mutated to cysteine, and truncated Dio3 (Dio3 , residues 3QFB) (32) using secondary structure matching in COOT (41). 120–304) was cloned in pET151 TOPO for expression in E. coli Rosetta2 cells, followed by nickel-nitrilotriacetic acid affinity chromatography, tobacco Blue-Native PAGE, Deiodinase Expression in COS-7 Cells, and Deiodinase etch virus protease digestion, and gel filtration. SeMet-labeled protein was Activity Assay. The protocol of Schägger et al. (46) was used for blue- expressed in SeMet medium (Molecular Dimensions) using E. coli strain B834 native PAGE. Expression plasmids carrying WT and mutant mouse Dio3 (DE3). Dio3cat crystals were set up at 20 °C with 20% (wt/vol) PEG 3350 and 0.2 M ammonium citrate (pH 7) as a reservoir solution. Diffraction datasets were cDNAs in pcDNA3 (Invitrogen) were transiently transfected in triplicate collected at 100 K at Swiss Light Source beamline X10SA for native Dio3cat and or stably transfected into COS-7 cells. The activity of 5-deiodinase was de- at European Synchrotron Radiation Facility ID14-4 for crystals of the SeMet- termined using a nonradioactive LC-tandem MS–based assay with direct de- 13 labeled protein. Indexing, scaling, and merging were done with HKL2000 (37) termination of rT3 in relation to an internal C-rT3 standard (47). and XDS (38) for native and SeMet crystals, respectively (Table 1). The structure of Dio3cat was solved by selenium single anomalous diffraction phasing using ACKNOWLEDGMENTS. We thank Barbara Kachholz, Anja Fischbach, Simone SHELXC, SHELXD, and SHELXE (39, 40) and refined using COOT (41) and Arndt, and the beamline staff for excellent technical support.

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