A [3Fe-4S] Cluster Is Required for Trna Thiolation in Archaea and Eukaryotes

A [3Fe-4S] cluster is required for tRNA thiolation in archaea and eukaryotes

Yuchen Liua,1, David J. Vinyardb,2, Megan E. Reesbeckb, Tateki Suzukic, Kasidet Manakongtreecheepc, Patrick L. Hollandb, Gary W. Brudvigb,c, and Dieter Söllb,c,1

aDepartment of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803; bDepartment of Chemistry, Yale University, New Haven, CT 06520; and cDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520

Contributed by Dieter Söll, September 23, 2016 (sent for review August 22, 2016; reviewed by Dennis R. Dean and David E. Graham) The sulfur-containing nucleosides in transfer RNA (tRNAs) are present significantly between different organisms. The bacterial s4U8 in all three domains of life; they have critical functions for accurate and and s2U34 biosynthetic pathways have mainly been studied in efficient translation, such as tRNA structure stabilization and proper Escherichia coli and Salmonella enterica, and they depend on codon recognition. The tRNA modification enzymes ThiI (in bacteria and persulfide group (R-S-SH) carriers for sulfur transfer (18). The archaea) and Ncs6 (in archaea and eukaryotic cytosols) catalyze the first step of both pathways is to derive the sulfur element from 4 2 formation of 4-thiouridine (s U) and 2-thiouridine (s U), respectively. free L-cysteine by cysteine desulfurases (IscS in E. coli), gener- The ThiI homologs were proposed to transfer sulfur via cysteine per- ating an initial protein-bound persulfide on an active site Cys sulfide enzyme adducts, whereas the reaction mechanism of Ncs6 residue in cysteine desulfurases (18). In E. coli, the persulfide of Meth- remains unknown. Here we show that ThiI from the archaeon IscS can then be directly transferred to ThiI that catalyzes s4U8 anococcus maripaludis contains a [3Fe-4S] cluster that is essential for its formation or indirectly transferred to the s2U34 formation en- tRNA thiolation activity. Furthermore, the archaeal and eukaryotic Ncs6 zyme MnmA through a sulfur relay chain composed of multiple homologs as well as phosphoseryl-tRNA (Sep-tRNA):Cys-tRNA synthase intermediate persulfide carriers (TusA, TusBCD complex, and (SepCysS), which catalyzes the Sep-tRNA to Cys-tRNA conversion in TusE) (2, 19). Both E. coli ThiI and MnmA contain a PP-loop methanogens, also possess a [3Fe-4S] cluster similar to the methanogenic archaeal ThiI. These results suggest that the diverse tRNA thio- motif and two active site Cys residues critical for activities (Fig. lation processes in archaea and eukaryotic cytosols share a common 1). The PP-loop binds ATP that is consumed to activate the mechanism dependent on a [3Fe-4S] cluster for sulfur transfer. target uridine by adenylation (Fig. 1A). The first catalytic Cys (Cys456 in E. coli ThiI and Cys199 in E. coli MnmA) receives the iron-sulfur cluster | thionucleosides | tRNA modification | CTU1 persulfide from IscS, and subsequently the ThiI- and MnmA- persulfide donate the sulfur to thiolate the activated C4 atom of ulfur atoms are present in several modified transfer RNA U8 and C2 atom of U34, respectively (20, 21). The second cat- S(tRNA) nucleosides, such as 4-thiouridine (s4U), 5-methyl-2- alytic Cys (Cys344 in E. coli ThiI and Cys102 in E. coli MnmA) is thiouridine derivatives (xm5s2U), 2-thiocytidine (s2C), and proposed to form a disulfide bond with the first Cys, assisting – 2-methylthioadenosine (ms2A) derivatives. The s4U nucleoside is sulfur release (20 22). The disulfide bond then needs to be re- found at position 8 of most bacterial and archaeal tRNAs and duced by a reductant before the next catalytic cycle. Notably, functions as a photosensor for near-UV irradiation (1). Upon many bacterial ThiI homologs lack the rhodanese homology irradiation, the s4U8 cross-links with the nearby cytidine at po- domain (RHD) (Fig. 1B) that contains the persulfide forming sition 13. This reaction causes conformational changes and prevents aminoacylation of tRNAs, resulting in accumula- Significance tions of uncharged tRNAs that trigger stringent responses (1). The

5 2 BIOCHEMISTRY xm s U nucleosides are present at position 34 (the wobble base) of Posttranscriptional transfer RNA (tRNA) modifications with Gln Glu Lys tRNA UUG,tRNA UUC,andtRNA UUU in all three domains sulfur atoms have important roles in accurate and efficient 5 2 of life. The rigid conformation of xm s U―preferentially in the translation and stress responses. Although the enzymes re- C3′-endo form―ensures efficient codon:anticodon base pairing sponsible for 2-thiouridine (s2U) and 4-thiouridine (s4U) modi- (1,2).Thepresenceofs2U in the codon:anticodon pair leading to a fications have been identified in archaea and eukaryotes, their preference for A-ending codons (3) may be explained by the greater catalytic mechanisms remain unclear because of the complexity stability of the s2U-A vs. s2U-G pair (4). Furthermore, the 2-thio of the sulfur transfer process. This study demonstrates that the group of xm5s2U acts as an identity element in aminoacylation re- sulfur insertion enzymes that catalyze the synthesis of s2Uin actions (5–7), promotes tRNA binding to the ribosomal A-site (7), archaea and eukaryotic cytosols, as well as s4U and Cys-tRNA and prevents frameshifting during translation (8). Yeast mutants in methanogenic archaea, all contain a [3Fe-4S] cluster. This lacking the 2-thio modification have pleotropic phenotypes, such as finding provides important insights into sulfur transfer re- defects in invasive growth (9), hypersensitivity to high temperature, action mechanisms and establishes a direct link between the rapamycin, caffeine, or oxidative stress (10, 11), inability to maintain ancient Fe-S cluster to translation in archaea and eukaryotes. normal metabolic cycles (12), and protein misfolding and aggregation (13). In humans, impaired 2-thio modification of the mitochondrial Author contributions: Y.L. and D.S. designed research; Y.L., D.J.V., M.E.R., T.S., and K.M. tRNAs has been associated with acute infantile liver failure (14, 15) performed research; Y.L., D.J.V., M.E.R., T.S., P.L.H., G.W.B., and D.S. analyzed data; and Y.L. and D.S. wrote the paper. and myoclonic epilepsy with ragged-red fibers (16, 17). Overall, s4U8 2 Reviewers: D.R.D., Virginia Polytechnic Institute; and D.E.G., Oak Ridge National and s U34 in tRNAs play important roles in translation. Laboratory. The sulfur transfer processes for s4U8 and s2U34 biosynthesis The authors declare no conflict of interest. are complicated, and some of the details (especially in archaea 1To whom correspondence may be addressed. Email: [email protected] or dieter.soll@ and eukaryotes) remain unclear because: (i) they usually involve yale.edu. a cascade of sulfur carrier proteins rather than a direct transfer 2Present address: Department of Biological Sciences, Louisiana State University, Baton from the ultimate sulfur donor to the substrate, (ii) some sulfur Rouge, LA 70803. carrier proteins have multiple roles that deliver sulfur to a variety This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of cofactors and nucleosides, and (iii) the sulfur carriers vary 1073/pnas.1615732113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1615732113 PNAS | November 8, 2016 | vol. 113 | no. 45 | 12703–12708 Downloaded by guest on October 1, 2021 Fig. 1. The reactions (A) and domain structures (B)ofs4U8 synthetase (ThiI), s2U34 synthetase (MnmA or Ncs6/Ncs2 complex), and SepCysS. (A) The AMP and phosphate moieties are highlighted in green, and the sulfur atoms in the products are highlighted in red. (B) The PP-loop motif and putative catalytic site Cys residues (with amino acid residue numbers indicated above) are colored in green and red, respectively. The domain structures of ThiI, MnmA, Ncs6, and SepCysS are based on the crystal structures of Bacillus anthracis ThiI (60), E. coli MnmA (21), Pyrococcus horikoshii TtuA (56), and Archaeoglobus fulgidus SepCysS (35), respectively.Abbreviations:Ado,adenosine;Ec, E. coli; Mmp, M. maripaludis; Sc, Saccharomyces cerevisiae; Mj, Methanocaldococcus jannaschii; NFLD, N-terminal ferredoxin-like domain; THUMP, thiouridine synthases, methylases and pseudouridine synthases; RHD, rhodanese homology domain; PLP, pyridoxal-5′-phosphate.

Cys (23, 24); therefore, the sulfur transfer mechanism of ThiI These findings suggest that both Ncs6 and the activated ubiq- without RHD remains unclear. uitin-like SAMP are required for s2U34 formation in archaea. The mechanism by which sulfur is incorporated into s2U34 in The archaeal s4U8 biosynthesis most likely resembles the eukaryotic cytosols differs greatly from that in bacteria. Initiated bacterial ThiI pathway because ThiI is widely distributed in ar- by Nfs1 (a cysteine desulfurase), the sulfur flow is directed chaea and deletion of the thiI gene in M. maripaludis eliminated through multiple persulfide carriers to a ubiquitin-related mod- s4UintRNAs(32).However,theE. coli s4U biosynthesis mecha- ifier (Urm1), generating a thiocarboxylate group on the carboxyl- nism cannot fully explain the archaeal process because the gene terminus of Urm1 (10, 25). This thiocarboxylate may be a sulfur encoding a cysteine desulfurase is missing in many sequenced ar- donor for s2U34 formation. The final step to activate and thio- chaeal genomes (23, 33) and most archaeal ThiI homologs lack the late the C2 atom of U34 is catalyzed by an enzyme complex— RHD essential to transfer sulfur. Similar to Ncs6, the methanogenic designated as Ncs6/Ncs2 in yeast, Ctu1/Ctu2 in nematode, and archaeal ThiI homologs have three conserved Cys residues (two ATPBD3/CTU2 in human (11, 25, 26)―in replacement of bac- from a CXXC motif) in the putative catalytic domain (Fig. 1B). A terial MnmA. Ncs6 has a PP-loop motif and three conserved Cys single mutation of any of the three Cys abolished M. maripaludis residues (two from a CXXC motif) in its putative catalytic do- ThiI activity (32), demonstrating that all three Cys residues are main (Fig. 1B). The PP-loop is presumably used for ATP binding crucial. This three-conserved Cys pattern is also present in Sep- to adenylate U34, resembling the reaction schemes of bacterial tRNA:Cys-tRNA synthase (SepCysS), which catalyzes tRNA- ThiI and MnmA; however, the sulfur transfer mechanism of dependent Cys biosynthesis in methanogenic archaea (Fig. 1) (34, Ncs6 is not known. Interestingly, the cytosolic Ncs6/Urm1 35). Mutational studies showed that the three Cys residues in pathway is dependent on the Fe-S cluster assembly machineries SepCysS are all critical for activity (36, 37). These findings imply (27), suggesting that it requires unidentified Fe-S proteins. that the three proteins―Ncs6 (in eukaryotes and archaea), ThiI (in The archaeal s2U34 biosynthetic pathway is proposed to re- methanogenic archaea), and SepCysS (in methanogenic archaea)― semble the eukaryotic cytosolic Ncs6/Urm1 pathway. This is have similar but unknown catalytic mechanisms dependent on three based on the observations that: (i) the ncs6 gene homologs are conserved Cys residues. Here we present evidence that these Cys widespread in archaeal genomes (23); (ii) deletion of the ncs6 residues coordinate a [3Fe-4S] cluster essential for tRNA thiolation. homolog (ncsA)inHaloferax volcanii resulted in only non- Lys thiolated tRNA UUU (28); (iii) Ncs6 homologs form complexes Results with the ubiquitin-like small archaeal modifier protein (SAMP), ThiI, SepCysS, and the Ncs6 Homolog in Methanogenic Archaea which has high structural homology to Urm1, in H. volcanii (29) Contain a [3Fe-4S] Cluster. The His6-tagged M. maripaludis ThiI and in Methanococcus maripaludis (30); and (iv) deletion of ei- (locus tag: MMP1354), the M. maripaludis Ncs6 homolog (locus ther samp2 or ubaA (encoding an E1-like protein that activates tag: MMP1356), and Methanocaldococcus jannaschii SepCysS Lys SAMPs) in H. volcanii eliminated thiolated tRNA UUU (31). (locus tag: MJ1678) proteins were recombinantly produced in

Fig. 2. UV-visible spectra of anoxically purified proteins in the as-purified (red) and after DTH reduced

(blue) states. The His6-tagged (A) M. maripaludis ThiI (330 μM), (B) M. maripaludis Ncs6 (540 μM), and (C) M. jannaschii SepCysS (670 μM) were produced and purified from E. coli.(Insets) The as-purified proteins were brownish in color; DTH reduction partially bleached the colors.

12704 | www.pnas.org/cgi/doi/10.1073/pnas.1615732113 Liu et al. Downloaded by guest on October 1, 2021 80 K displayed one quadrupole doublet with an isomer shift δ = −1 −1 0.28 mm·s and a quadrupole splitting jΔEQj = 0.63 mm·s (Fig. + + 3B). These values indicate a [3Fe-4S]1 cluster with all Fe3 ions (39). After reduction with 5 mM DTH, the spectrum was best fit to two quadrupole doublets in a 2:1 ratio (Fig. 3B).Themoreintense −1 −1 doublet with δ = 0.49 mm·s and jΔEQj = 1.08 mm·s corresponds 2.5+ −1 to an (Fe )2 pair, and the less intense doublet with δ = 0.35 mm·s −1 3+ and jΔEQj = 1.13 mm·s corresponds to an Fe site (Fig. 3B). These values agree with a reduced [3Fe-4S]0. Collectively, the EPR and Mössbauer spectra indicate that the as-purified proteins harbor + an oxidized [3Fe-4S]1 cluster that can be completely reduced to [3Fe-4S]0 with DTH. To verify that the recombinant protein produced in E. coli has the same cluster as the one in archaea, the Strep-tagged M. maripaludis ThiI was produced and anoxically purified from its natural host M. maripaludis. The as-purified protein contained 2.9 ± 0.1 Fe per protomer and exhibited identical features in the UV-visible (Fig. S1A) and EPR (Fig. S1B and Table S1) spectra compared with the protein purified from E. coli. These results suggest that M. maripaludis ThiI contains a [3Fe-4S] cluster irrespective to our tested expression hosts. Fig. 3. EPR and Mössbauer spectra of anoxically purified proteins. (A) The X-band EPR spectra of M. maripaludis ThiI (530 μM), M. maripaludis Ncs6 Eukaryotic Ncs6 Contains a [3Fe-4S] Cluster. The Saccharomyces cer- (540 μM), and M. jannaschii SepCysS (670 μM) in the as-purified (red) and evisiae Ncs6 and its human homolog (designated as CTU1 or DTH reduced (blue) states. The experimental conditions are as follows: mi- ATPBD3) share ∼30% sequence identity with the M. maripaludis crowave power, 2 μWforM. maripaludis ThiI, 200 μWforM. maripaludis Ncs6 homolog. To characterize the eukaryotic protein, the maltose μ Ncs6, 100 WforM. jannaschii SepCysS; microwave frequency, 9.4 GHz; modula- binding protein (MBP)-tagged human CTU1 was produced in tion amplitude, 10 G; temperature, 7K. (B) The zero-field 57Fe Mössbauer spectra of μ E. coli and purified under anoxic conditions. The as-purified protein the as-purified (Upper) and DTH reduced (Lower) M. maripaludis ThiI (180 M). The ± experimental data (gray dots) were recorded at 80 K. (Upper) The best fit contained 1.0 0.1 Fe per protomer and exhibited an absorption − − quadrupole doublet (red trace) has δ = 0.28 mm·s 1 and jΔE j= 0.63 mm·s 1. peak around 420 nm in the UV-visible spectrum (Fig. 4A). Fur- Q 57 (Lower) The best fit (red trace) is a sum of two quadrupole doublets: the thermore, the Fe Mössbauer spectrum of the as-purified human −1 −1 major component with δ = 0.49 mm·s and jΔEQj= 1.08 mm·s (green) and the CTU1 recorded at 80 K displayed one quadrupole doublet with δ = δ = · −1 jΔ j = · −1 −1 −1 minor component with 0.35 mm s and EQ 1.13 mm s (blue). 0.29 mm·s and jΔEQj = 0.58 mm·s , consistent with the signal of δ = · −1 jΔ j = 1+ About 5% impurity is not included in the fit with 0.71 mm s and EQ an oxidized [3Fe-4S] cluster (Fig. 4B). These results suggest that · −1 2+ 3.04 mm s indicating an Fe species. the eukaryotic cytosolic Ncs6 has a [3Fe-4S] cluster similar to that in archaeal proteins. E. coli and purified under anoxic conditions. The purified pro- The [3Fe-4S] Cluster Is Essential for tRNA Thiolation Activity. The im- teins were brownish in color (Fig. 2, Insets), exhibiting a broad portance of the Fe-S cluster for M. maripaludis ThiI enzyme activity band at around 420 nm in UV-visible absorption spectra (Fig. 2). was examined using [(N-acryloylamino)phenyl] mercuric chloride Addition of 5 mM sodium dithionite (DTH) partially bleached the (APM) gel electrophoresis, a method that detects sulfur modifica- protein color and decreased the UV-visible absorption. Chemical ± tions in RNAs (40). Cysteine, thiosulfate, thiophosphate, and sul- analysis of Fe content indicated that the proteins contained 2.8 fide were previously tested as possible sulfur sources, but only

± BIOCHEMISTRY 0.2 Fe per protomer of M. maripaludis ThiI, 2.7 0.3 Fe per pro- sulfide supported the formation of thiolated tRNA exhibiting re- tomer of M. maripaludis Ncs6, and 1.2 ± 0.5 Fe per protomer of tarded migration in APM gels (32). The KM of Na2S(∼1mM)(32) M. jannaschii SepCysS. When any of the three conserved Cys was is close to the estimated intracellular concentrations of free sulfide altered to Ala (C265, C268, and C348 in MMP1354; C142, C145, in methanococci (∼1–3 mM) (41), suggesting that sulfide is a C233 in MMP1356; C64, C67, and C272 in MJ1678), the proteins physiologically relevant sulfur donor. In this study, the anoxically < were colorless and contained no detectable Fe ( 0.1 Fe per pro- purified M. maripaludis ThiI, which contained a [3Fe-4S] cluster, tomer). These spectral properties and the presence of substantial was active to produce thiolated tRNA when using M. jannaschii amounts of associated Fe in wild-type proteins suggest that they contain Fe-S clusters coordinated by three conserved Cys residues. To confirm the presence of an Fe-S cluster and determine the cluster type, we used electron paramagnetic resonance (EPR) and Mössbauer spectroscopies. The EPR spectra of all three as-purified (following affinity purification steps inside an anaerobic chamber without addition of a reducing agent) proteins measured at 7 K displayed a peak centered at gav = 2.00 (Fig. 3A and Table S1), 1+ characteristic of a cuboidal [3Fe-4S] cluster (Stol = 1/2) containing + three Fe3 ions (38). After treatment with 5 mM DTH, all proteins were EPR-silent in perpendicular-mode measurement, consistent with a one-electron reduction of the cluster to the high spin 0 3+ [3Fe-4S] state (Stot = 2) resulting from an Fe ion and a mixed- + valent (Fe2.5 ) pair (38). Attempts to reoxidize the cluster in Fig. 4. Spectroscopic characterization of the recombinant MBP-tagged 2 human CTU1. (A) UV-visible spectra of anoxically purified protein (420 μM) M. maripaludis ThiI by treatment with 2 mM potassium ferricyanide in the as-purified (red) and after DTH reduced (blue) states. (B) The zero-field or by exposure to air were unsuccessful, resulting in precipitated 57Fe Mössbauer spectrum of the as-purified CTU1 (420 μM). The experi- 57 protein and no significant EPR signal. The zero-field Fe Möss- mental data (gray dots) were recorded at 80 K and fit to one quadrupole −1 −1 bauer spectrum of the as-purified M. maripaludis ThiI recorded at doublet with δ = 0.29 mm·s and jΔEQj = 0.58 mm·s (red trace).

Liu et al. PNAS | November 8, 2016 | vol. 113 | no. 45 | 12705 Downloaded by guest on October 1, 2021 Cys 1+ tRNA ,Na2S, and ATP as the substrates (Fig. 5A). When 5 μMof Na2S. One possibility is that Na2S partially reduced the [3Fe-4S] as-purified M. maripaludis ThiI was used in this assay, about 18 μM cluster to the EPR-silent [3Fe-4S]0 state. In agreement with the of M. jannaschii tRNACys was thiolated according to band in- reduction, the zero-field 57Fe Mössbauer spectrum of M. maripaludis tensities (Fig. 5F); this finding suggests that M. maripaludis ThiI can ThiI incubated with Na2S was best fit to the sum of two quadrupole − catalyze multiple turnovers using exogenous sulfide. On the other doublets in a 1:1 ratio (Fig. 5E). One doublet with δ = 0.43 mm·s 1 −1 2.5+ hand, after incubation with 10mMEDTAinthepresenceof and jΔEQj = 1.14 mm·s corresponds to Fe ,andtheother −1 −1 10 mM DTH, M. maripaludis ThiI became colorless and lost its doublet with δ = 0.30 mm·s and jΔEQj = 0.60 mm·s corresponds + cluster (<0.1 Fe per protomer). This apo-protein was inactive to to Fe3 (Fig. 5E); this spectrum suggests a mixture of 25% of 1+ 0 produce thiolated tRNA (Fig. 5A), indicating that the Fe-S cluster is [3Fe-4S] and 75% of [3Fe-4S] after incubation with Na2S. To required for the tRNA thiolation activity of M. maripaludis ThiI. further investigate whether the reduced state is active, we tested the To reactivate the apo-protein, it was incubated with excess DTH-treated M. maripaludis ThiI containing only the reduced + amounts of Fe2 and sulfide in the presence of DTT. This treat- cluster. This completely reduced enzyme showed similar activity to ment resulted in brownish-colored protein, showing a broad band at the as-purified protein containing the oxidized cluster (Fig. 5F). around 420 nm in the UV-visible absorption spectrum similar to the These observations suggest that the enzyme can catalyze the thio- as-purified protein (Fig. 5B). The EPR spectrum displayed a peak lation reaction irrespective of the initial redox state of the cluster, centered at gav = 2.01 (Fig. 5C and Table S1), demonstrating the although we cannot eliminate the possibility that the initially oxidized 1+ successful reconstitution of an oxidized [3Fe-4S] cluster. The cluster needs to be reduced by Na2S for activity. Furthermore, the chemical analysis of Fe content agreed with the spin quantification reduced enzyme was unable to produce thiolated tRNA without ∼ of the EPR signal, indicating that 50% of the total protein turned Na2S(Fig.5F), suggesting that Na2Sisrequiredasanexogenoussulfur into the holo-form after reconstitution. As expected, the in vitro source rather than a reductant under this experimental condition. reconstituted cluster restored the tRNA thiolation activity of M. maripaludis ThiI (Fig. 5A). Discussion To investigate the active form of the cluster during reaction, Taking these data together, we demonstrate by UV-visible absor- M. maripaludis ThiI was incubated with the substrates (Na2Sor bance, EPR, and Mössbauer spectroscopies that the archaeal and ATP + tRNACys) and monitored by EPR spectroscopy. None of eukaryotic Ncs6 homologs as well as the methanogenic archaeal the substrates altered the gav (Fig. 5D), suggesting that the ThiI and SepCysS are all [3Fe-4S] cluster-containing enzymes. + cluster type at least partially remained as [3Fe-4S]1 upon sub- Despite lacking significant sequence homology, these three proteins strate binding. In comparison with the as-purified protein, the all have three conserved Cys residues (two from a CXXC motif) in spin quantification of the EPR signal was not significantly their putative catalytic domains. A single mutation of any of the changed with the addition of ATP + tRNACys but decreased with three Cys removed protein-bound Fe, suggesting that these Cys

Fig. 5. The [3Fe-4S] cluster in M. maripaludis ThiI is essential for the tRNA thiolation activity. (A)The tRNA thiolation activity of the as-purified (Left), EDTA-treated (Center), and in vitro reconstituted M. maripaludis ThiI (Right) was analyzed with the APM-retardation gel electrophoresis assay. The protein (1 μM) was incubated with the unmodified M. jannaschii tRNACys transcript (20 μM) together

with 2 mM ATP and 5 mM Na2S for 0, 5, 15, 30, or 60 min. The relative positions of unmodified and thiolated tRNAs are labeled. (B) The UV-visible spectra of EDTA-treated (gray) and reconstituted (red) M. maripaludis ThiI. (C) The X-band EPR spectrum of the reconstituted M. maripaludis ThiI recorded at microwave power, 20 μW; microwave frequency, 9.4 GHz; modulation amplitude, 10 G; temperature, 9 K. (D) The X-band EPR spectra of the as-purified M. maripaludis ThiI (280 μM) incubated with 5 mM Cys Na2S(Upper) or 2 mM ATP + 200 μM tRNA (Lower) recorded at microwave power, 20 μW; microwave frequency, 9.4 GHz; modulation amplitude, 10 G; temperature, 7 K. (E) The zero-field 57Fe Mössbauer spectra of the as-purified M. maripaludis ThiI (340 μM)

incubated with 5 mM Na2S. The experimental data (gray dots) were recorded at 80 K. The best fit (red trace) is a sum of two quadrupole doublets―one with −1 −1 δ = 0.43 mm·s and jΔEQj= 1.14 mm·s (blue) and the −1 −1 other one with δ = 0.30 mm·s and jΔEQj= 0.60 mm·s (green)―in a 1:1 ratio. (F) The tRNA thiolation activity of as-purified M. maripaludis ThiI (5 μM) reacted with the unmodified M. jannaschii tRNACys transcript (20 μM),

2mMATP,and5mMNa2S(Left), 5 mM DTH (Center), or 5 mM Na2S + 5mMDTH(Right).

12706 | www.pnas.org/cgi/doi/10.1073/pnas.1615732113 Liu et al. Downloaded by guest on October 1, 2021 residues chelate the Fe-S cluster. A trisulfide linkage was previously the putative catalytic domain are essential for TtuA activity (56). detected in the CXXC motif by mass spectrometry analyses of M. Although the solved crystal structure of TtuA did not show the maripaludis ThiI, M. maripaludis Ncs6 homolog, and M. jannaschii presence of a cluster, the same requirement of Cys residues in SepCysS (30, 32, 36); however, the sulfane sulfur (S0) carried by Cys Ncs6 and TtuA suggests that TtuA may also have an Fe-S cluster may come from oxidation of an Fe-S cluster, which was similarly coordinated by the three critical Cys residues. In this scenario, observed upon oxidative decomposition of a spinach ferredoxin (42) the requirement of an Fe-S cluster is a common feature of the and aconitase (43). The three conserved Cys residues are essential TtcA/TtuA family that is used for tRNA thiolation by all three for in vivo activities of M. maripaludis ThiI (32) and M. jannaschii domains of life. SepCysS (36). Similarly, the corresponding CXXC motif is required for the Ncs6 activity in yeast (11). In this study, we reveal that the Materials and Methods M. maripaludis M. maripaludis [3Fe-4S] cluster is indispensable for the tRNA thiolation activity of Production and Anoxic Purification of His6-Tagged ThiI, M. maripaludis ThiI; this suggests that the critical role of these Ncs6, and M. jannaschii SepCysS. The M. maripaludis thiI (locus tag: MMP1354), conserved Cys residues is Fe-S cluster coordination. Overall, the M. maripaludis ncs6 (locus tag: MMP1356), and M. jannaschii pscS (locus tag: same essential Cys pattern and cluster type discovered in ThiI, Nsc6, MJ1678) genes were cloned into the pQE2 (with an N-terminal His6-tag), pDCH (with a C-terminal His6-tag), and pET15b (with an N-terminal His6-tag) vectors, and SepCysS suggest that they have a common reaction mechanism respectively. For expression, the vectors were transformed into the E. coli Rosetta dependent on a [3Fe-4S] cluster. This finding now explains why 2 2(DE3) strain (Novagen). The details of protein production and purification are in previous genetic studies showed that the biosynthesis of s Uin SI Materials and Methods. eukaryotic cytosolic tRNAs requires the Fe-S cluster assembly machinery (27). Production and Anoxic Purification of Strep-Tagged M. maripaludis ThiI from The reaction mechanism of a [3Fe-4S] cluster-dependent thi- M. maripaludis. The M. maripaludis thiI gene (locus tag: MMP1354) was olation remains to be explored. The [3Fe-4S] clusters are less cloned into the pAW42 vector with an N-terminal Strep-tag (WSHPQFEK) common than the cubic [4Fe-4S] clusters in natural proteins. A and transformed into the M. maripaludis strain S0001 for expression. The few cases of [3Fe-4S]-containing proteins include [NiFe] hy- details of culture condition, transformation, expression, and protein purifi- drogenase (44), succinate dehydrogenase (45), and nitrate re- cation are in SI Materials and Methods. ductase (46), in all of which the [3Fe-4S] clusters have been Production and Anoxic Purification of MBP-Tagged ATPBD3. The codon opti- proposed to transfer electrons. We propose three thoughts of the mized Homo sapiens gene ATPBD3 (CTU1) was cloned into the pMA-c5x role of the [3Fe-4S] clusters in tRNA thiolation reactions. vector (New England Biolabs) with an N-terminal MBP-tag. The expression (i) The cluster is possibly required for binding to a substrate such vectors were transformed into the E. coli BL21(DE3) strain. The details of as tRNA, ATP, or an adenylated intermediate. For example, the protein production and purification are in SI Materials and Methods. [4Fe-4S] clusters in E. coli RlmD/RumA (47) and Thermotoga maritima tryptophanyl-tRNA synthetase (TrpRS) (48) are pro- Analytical and Spectroscopic Measurements. All analytical analyses were posed to be involved in RNA substrate recognition but not di- performed in triplicate. Protein concentrations were determined using the rectly in catalysis. (ii) The cluster may be a direct sulfur donor, BCA Protein Assay Kit (Pierce). Iron was quantified by using the Quantichrom providing a sulfur atom that is inserted to the substrate. For Iron Assay Kit (BioAssay Systems). UV-visible absorption spectra were example, a [2Fe-2S] cluster in biotin synthase has been proposed recorded on a Nanodrop 2000c spectrometer with samples in quartz cuvettes (optic path = 1 cm) closed with rubber stoppers under anoxic conditions. to donate a sulfur atom into the biotin thiophane ring; the cluster is X-band EPR spectra were recorded at 7–9 K on a Bruker ELEXSYS E500 killed during the reaction (49). To explain that exogenous Na2Sis spectrometer equipped with a SHQ resonator and Oxford ESR-900 helium flow required for the M. maripaludis ThiI reaction and M. maripaludis cryostat. Multiple microwave powers were tested so that resonances were ThiI can have multiple turnovers, the [3Fe-4S] cluster needs to be quantified under nonsaturating conditions. The g values (Table S1) were de- regenerated from sulfide during catalysis. (iii) The cluster may be termined by simulating spectra using EasySpin 5.0.5 (57). Mössbauer spectra responsible for sulfur transfer by ligating to an activated sulfur spe- were recorded at 80 K on a SeeCo Mössbauer spectrometer with alternating cies. For example, the methylthiotransferases MiaB, which catalyzes acceleration and a 0.07 mT magnetic field. The isomer shifts were referenced to the conversion of i6Atoms2i6A in tRNAs, carries a polysulfide iron metal at 298 K. Sample temperature was maintained at 80 K with a Janis bridge connecting its two [4Fe-4S] clusters (50). This polysulfide is Research Company cryostat. The spectra were simulated and fit using WMoss BIOCHEMISTRY (SeeCo) with Lorentzian doublets. The fits are anisotropic as a result of the likely derived from an exogenous sulfur source and used as the sulfur clusters’ paramagnetism affecting the relaxation time of the transitions (58, 59). donor to generate ms2i6A. Similar to the proposed MiaB reaction mechanism, an exogenous sulfur may be ligated to an Fe ion of the In Vitro Reconstitution of Fe-S Cluster and Enzymatic Assay. All following steps [3Fe-4S] cluster in Nsc6, ThiI, and SepCysS and then transferred to were performed under anoxic conditions. To prepare apo-protein, the purified their substrates. The Fe ion, when ligated to an activated sulfur M. maripaludis ThiI was treated with 10 mM EDTA and 10 mM sodium DTH at species, may dissociate from its Cys residue ligand or still link to the 4 °C for 4 h. The apo-protein was buffer-exchanged using a PD MiniTrap G-25 residue as a Cys persulfide ligand (51). column (GE Healthcare) pre-equilibrated with 50 mM sodium Hepes (pH 7.5), The Ncs6 sequence is most closely related to TtuA and TtcA, 0.3 M NaCl, and 25% (vol/vol) glycerol. To reconstitute the Fe-S cluster, the belonging to the TtcA/TtuA family. TtuA catalyzes the bio- protein was first incubated with 5 mM DTT at room temperature for 1 h. Then a synthesis of 2-thioribothymidine (s2T) at position 54 in thermo- 10-fold molar excess of Na2S followed by an 8-fold molar excess of Fe(NH4)2(SO4)2 was added. The mixture was incubated at room temperature for 1 h or at 4 °C philic bacterial and archaeal tRNAs (52, 53), whereas TtcA 2 overnight. The excess reagents were then removed by passing through a PD catalyzes s C formation at position 32 in bacterial and archaeal MiniTrap G-25 column (GE Healthcare) pre-equilibrated with 50 mM sodium tRNAs (54). The TtcA sequence has two CXXC motifs, and a Hepes (pH 7.5) and 0.3 M NaCl. After concentrating with an Amicon Ultra cen- recent study of E. coli TtcA showed that this protein possesses a trifugal filter (30-kDa molecular weight cut-off), the reconstituted protein was [4Fe-4S] cluster that is crucial for the thiolation activity (55). supplemented with glycerol [final concentration 30% (vol/vol)]. Although the reaction mechanism remains unclear, the [4Fe-4S] The in vitro tRNA thiolation reaction catalyzed by M. maripaludis ThiI was cluster is suggested to coordinate a sulfide (–SH) group that is performed under anoxic conditions as described (32). ATP (final concentration Cys the proximal sulfur donor to generate s2C (55). In contrast, both 2mM),M. jannaschii tRNA transcript (final concentration 20 μM), and freshly Ncs6 and TtuA contain five CXXC(H) motifs, which are char- prepared Na2S (final concentration 5 mM) were added to the as-purified, EDTA- treated, or cluster reconstituted M. maripaludis ThiI (1 μM) to initiate the re- acteristics of the group II TtcA family (54). A crystal structure of action. To observe time-dependence, 50-μL reactions in buffer containing 50 mM TtuA showed that TtuA has two Zn finger domains (each with sodium Hepes (pH 7.5), 0.3 M NaCl, 5 mM MgCl2 was carried out at 37 °C for two CXXC/H motifs) and a putative catalytic domain (with a PP- 5–60 min. At each time point, 6 μL aliquots were mixed with 6 μL formamide loop motif, a CXXC motif, and an additional conserved Cys) loading dye to quench the reaction. The mixture was analyzed by APM- (56). Mutational analyses revealed that the three Cys residues in retardation gel electrophoresis as described previously (32). The relative

Liu et al. PNAS | November 8, 2016 | vol. 113 | no. 45 | 12707 Downloaded by guest on October 1, 2021 positions of unmodified and thiolated tRNACys were compared with those the National Science Foundation Grant MCB-1410079 (to Y.L.); National In- reported previously (32). stitute of General Medical Sciences Grants GM22854 (to D.S.) and GM065313 (to P.L.H.); and US Department of Energy, Office of Science, Office of Basic ACKNOWLEDGMENTS. We thank Drs. Michael Johnson, Evert Duin, and Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences Akiyoshi Nakamura for insightful discussions. This work was supported by Grant DE-FG02-05ER15646 (to G.W.B.).

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