Formation of a Ternary Complex for Selenocysteine Biosynthesis in Bacteria*

Background: Selenoprotein biosynthesis requires the interaction of tRNASec and specific enzymes that drive the synthesis of selenocysteine. Results: Formation of a molecular complex of selenophosphate synthetase, selenocysteine synthase, and tRNASec was identified and characterized. Conclusion: The ternary complex formation is necessary for selenoprotein synthesis. Significance: Our findings demonstrate the formation of a ternary complex and provide a possible scenario for selenium metabolism in bacteria. The synthesis of selenocysteine-containing proteins (selenoproteins) involves the interaction of selenocysteine synthase (SelA), tRNA (tRNASec), selenophosphate synthetase (SelD, SPS), a specific elongation factor (SelB), and a specific mRNA sequence known as selenocysteine insertion sequence (SECIS). Because selenium compounds are highly toxic in the cellular environment, the association of selenium with proteins throughout its metabolism is essential for cell survival. In this study, we demonstrate the interaction of SPS with the SelA-tRNASec complex, resulting in a 1.3-MDa ternary complex of 27.0 ± 0.5 nm in diameter and 4.02 ± 0.05 nm in height. To assemble the ternary complex, SPS undergoes a conformational change. We demonstrated that the glycine-rich N-terminal region of SPS is crucial for the SelA-tRNASec-SPS interaction and selenoprotein biosynthesis, as revealed by functional complementation experiments. Taken together, our results provide new insights into selenoprotein biosynthesis, demonstrating for the first time the formation of the functional ternary SelA-tRNASec-SPS complex. We propose that this complex is necessary for proper selenocysteine synthesis and may be involved in avoiding the cellular toxicity of selenium compounds.

high chemical reactivity of its metabolites (1,2). Organisms in all three domains of life (bacteria, archaea, and eukarya) synthesize selenocysteine (Sec) 3 as the main form of organic selenium in the cells, which is incorporated into specialized proteins, known as selenoproteins, that are involved in several functions including oxidoreductions, redox signaling, and antioxidant defense (1,3).
Sec is synthesized on the specific L-serine-aminoacylated tRNA (Ser-tRNA Sec ) and incorporated into selenoproteins at UGA codons via a complex pathway that works through transient protein-RNA and protein-protein interactions. In bacteria, this pathway requires the specific tRNA Sec (SelC) and an mRNA-specific structure called selenocysteine insertion sequence (SECIS) (1,3). Escherichia coli tRNA Sec has 8-and 5-bp stems in the acceptor and T arms, respectively, whereas the canonical tRNAs have a 7ϩ5 secondary structure. The D arm of E. coli tRNA Sec has a 6-bp stem and a 4-nucleotide loop, whereas the canonical tRNAs have a 3-4-bp D stem and 7-12nucleotide D loop. In addition, the extra arms of the bacterial tRNA Ser have 5-7-bp stems, in contrast to the 6 -9-bp stem observed in E. coli tRNA Sec (4).
Sec biosynthesis is initiated by the conversion of L-seryl-tRNA Sec , aminoacylated with serine by seryl-tRNA synthetase (SerRS), to L-selenocysteyl-tRNA Sec in a reaction catalyzed by selenocysteine synthase (E.C. 2.9.1.1., SelA), which is a pyridoxal 5Ј-phosphate (PLP)-dependent homodecameric enzyme of ϳ500 kDa (5). The co-factor PLP is covalently linked to the Lys 295 amino acid residue in each monomer of E. coli SelA prior to Ser-Sec conversion (5). Therefore, seryl-tRNA Sec is linked to SelA in the cofactor site, resulting in a binary complex consisting of one SelA decamer :10 tRNA Sec (6). Recently, the structures of Aquifex aeolicus SelA and its binary complex SelA-tRNA Sec were resolved by x-ray crystallography, highlighting that the decameric conformation is mandatory to provide the catalytic site for binding the tRNA molecule (4).
To achieve Ser-Sec conversion, selenium is transferred to the binary complex on its biologically active form, selenophosphate, a product of the reaction catalyzed by the 72.4-kDa dimeric enzyme selenophosphate synthetase (E.C. 2.7.9.3, SelD or SPS), from selenide and ATP (8). Selenophosphate is produced in a two-step reaction, in which selenide is phosphorylated by the ATP ␥-phosphate moiety and then ADP is hydrolyzed, releasing selenophosphate, AMP, and orthophosphate (8 -11). Selenide originates from selenite reduction, from converted methylated selenium compounds, or through selenium removal from selenoprotein degradation (12).
Because the K m value of 20 M for selenide in vitro results in toxic levels of this compound in the cellular environment, it was hypothesized that SPS in vivo obtains selenide from the PLPdependent NifS-like enzymes CsdB, CSD, and IscS (12). In E. coli, these PLP-donor enzymes act as ␤-lyases, catalyzing the cleavage of the C-S bond from Cys or the C-Se bond from Sec to Ala and S 0 or Se 0 , respectively (3,11,13). However, an interaction between SPS and NifS-like enzymes has not been described, although a structural basis for the interaction of E. coli CsdB and A. aeolicus SPS was proposed because the molecular surfaces surrounding the active sites of CsdB and SPS exhibit complementarity by molecular docking (10). It is possible that thioredoxin reductase, which is involved in selenite reduction, is also involved in delivering selenide for SPS (3,13). After selenophosphate is synthesized, it remains bound to the active-site cavity of SPS until ADP hydrolysis occurs and the product release is completed (7, 10).
Itoh et al. (10) hypothesized that SPS could interact with SelA in a manner similar to that of NifS-like proteins, facilitating the efficient transfer of selenophosphate from SPS to SelA; however, this interaction has never been formally proven. Interestingly, the human SepSecS was reported to interact in vivo with the SPS1 isoform (14), but little is known about the mechanism of this interaction. The elucidation of SPS-catalyzed selenium metabolism is important because SPS, rather than the less specific SelA, is responsible for the discrimination between selenium and sulfur in the process of Sec-tRNA Sec biosynthesis. The structural basis for this specificity is not yet understood.
In this study, we show that SPS functionally interacts with the SelA-tRNA Sec binary complex, forming the SelA-tRNA Sec -SPS complex. The macromolecular assembly of the ternary complex follows a stoichiometric ratio of 1SelA decamer :10tRNA Sec : 5SPS dimer , resulting in a macromolecular structure of ϳ1.3 MDa, and we provide structural insights into the organization of the ternary complex.

Experimental Procedures
Expression and Purification of E. coli SelA, ⌬28-SelA, SPS, and ⌬11-SPS-SelA was expressed and purified according to Manzine et al. (15) in binding buffer consisting of 20 mM potassium phosphate (pH 7.5), 100 mM sodium chloride, 5% glycerol, 2 mM ␤-mercaptoethanol, and 10 M PLP. The ⌬28-SelA truncated N-terminal domain was amplified from selA-pET29a vector using 5Ј-CATATGGCTATTGATCGCTTATTG-3Ј forward and 5Ј-GCGGCCGCTCATTTCAACAACATCTCC-3Ј reverse primers and then ligated into the same vector used by Manzine et al. (15) and transformed into the selA(Ϫ) E. coli strain JS1. The DNA sequence of E. coli SPS was amplified from E. coli genomic DNA using 5Ј-ACTGTATCATATGAGCGA-GAACTCGATTCGTTTGACCCAATAC-3Ј forward and 5Ј-TGCACTCGAGTCATTAACGAATCTCAACCATGGCAC-GACCGAC-3Ј reverse primers and ligated into pET28a(ϩ) vector (GE Healthcare). Recombinant SPS was overexpressed at 37°C overnight in the E. coli BL21 (DE3) (Stratagene) in LB medium and then harvested at 12,000 ϫ g for 15 min at 4°C. The pellet was resuspended in buffer A (50 mM Tris/HCl, pH 8.0, 10 mM imidazole, 300 mM NaCl) and lysed by six cycles of 30 s of sonication and 1 min of rest using the 550 Sonic Dismembrator (Fisher Scientific). The soluble fraction was applied to a metal-chelate affinity matrix (nickel-nitrilotriacetic acid, Qiagen) and eluted with 250 mM imidazole, followed by cleavage of the affinity tag using 1 unit of thrombin protease (GE Healthcare) for 100 g of E. coli SPS. The product was purified to homogeneity using size exclusion chromatography (Superdex 200, GE Healthcare) in 50 mM Tris/HCl buffer, pH 8.0, 300 mM NaCl, and 5 mM DTT. Limited proteolysis of E. coli SPS was performed using chymotrypsin protease (Sigma). SPS (5 mg/ml) was incubated at a protease:protein ratio of 1:50 w/w for 20 min at 18°C and analyzed by SDS-PAGE. A stable proteolytic fraction was subjected to N-terminal sequencing by Edman degradation (Department of Biochemistry, University of Cambridge). The result from the proteolytic digestion was used to confirm the truncation of the N-terminal sequence of E. coli SPS after the 11th amino acid residue. The ⌬11-SPS construct lacking the first 11 amino acid residues was obtained by DNA sequence amplification from E. coli genomic DNA using 5Ј-AGCATATGAGCCACGGAGCTGGTTGCGGCTG-3Ј forward and 5Ј-AGCTCGAGTTAACGGATCTCAACCATG-GCACG-3Ј reverse primers and ligated into pET28a(ϩ) vector (GE Healthcare).
Cloning of E. coli tRNA Sec , in Vitro Transcription, and Fluorescein-labeled tRNA Sec and tRNA Sec Mutant Constructs-We used the protocol described by Manzine et al. (6) to obtain the E. coli tRNA Sec (5). For fluorescence spectroscopy assays, E. coli tRNA Sec was labeled with fluorescein maleimide using the 5Ј EndTag TM nucleic acid labeling system (Vector Laboratories, Burlingame, CA) according to Manzine et al. (6).  (6) Functional Complementation Assay-The functional complementation experiments were conducted according to Sculaccio et al. (16) for N-terminally truncated SPS. Briefly, the E. coli strain WL400 (DE3), which lacks the functional selD gene (7), was transformed with the full-length E. coli SPS sequence and the SPS construct lacking the N-terminal 11 residues (⌬11-SPS). These cells were tested for the presence of an active selenoprotein formate dehydrogenase H (FDH H) using the benzyl viologen assay under anaerobic conditions (16). Similarly, the SelA and N-terminally truncated SelA complementation experiments were performed using this methodology using the E. coli strain JS1 (DE3), which lacks the functional selA gene, under the same anaerobic conditions (16) for 48 h in 30°C.
Fluorescence Anisotropy Assay-Fluorescence anisotropy measurements were performed in an ISS-PC spectrofluorometer (ISS, Champaign, IL). The uncharged tRNA Sec was fluorescein-labeled, and its interaction with SelA was conducted using 500 nM SelA with 490 nM unlabeled tRNA Sec and 10 nM fluorescein-labeled tRNA Sec incubated in binding buffer for 30 min at 25°C to form the covalently bound binary complex SelA-tRNA Sec in a final equimolar stoichiometry, according to previous publications (5,6). The isothermal fluorescence anisotropy assay was performed with fluorescence anisotropy measurements in "L" geometry at 25°C. A concentrated SPS sample was titrated to a SelA-tRNA Sec sample, homogenized, and equilibrated for 5 min at 25°C prior to steady-state anisotropy measurements. The same experimental conditions were applied to fluorescence anisotropy assays using mutant tRNA Sec constructs. Excitation was set to 480 nm, and emission was recorded through an orange cut-off filter at 515 nm (6). Anisotropy fluorescence values, r, and total intensity of fluorescence were calculated with the ISS program. In all cases, maximal dilution was less than 20%. The resulting fluorescence anisotropy values were fitted, using the program Origin 8.0, to the Hill equation with r 0 and r f representing the initial and final fluorescence anisotropy measures. [SPS monomer ] is the titrated SPS concentration in units of monomers. Thus, the apparent dissociation constant (K d ) and the Hill constant (n) were determined.
Experiments for determination of the stoichiometry of SelA-tRNA Sec -SPS binding were performed using 5000 nM SelA bound to 4990 nM unlabeled tRNA Sec and 10 nM fluoresceinlabeled tRNA Sec . The same procedures as described above were used during the SPS titration. Mutant tRNA Sec molecules were also tested for interaction with SelA by fluorescence anisotropy assays, as described previously (6).
Hydrogen/Deuterium Exchange Analyzed by Mass Spectrometry (H/DEx-MS)-We used hydrogen/deuterium exchange coupled with mass spectrometry to map the surfaces of SelA and SPS following the formation of the SelA-tRNA Sec binary complex and the SelA-tRNA Sec -SPS ternary complex. The various samples (SelA, SelA-tRNA Sec , SPS, tRNA Sec , and SelA-tRNA Sec -SPS) were prepared using a published protocol (17). Briefly, the samples were labeled by diluting the sample to a final concentration of D 2 O of ϳ90%. At each time point analyzed, aliquots (20 l) were taken out of the exchange tube and quenched by mixing the solution with a 1:1 ratio of the quenching buffer (D 2 O, 100 mM sodium phosphate, pH 2.5) and cooled to 0°C to slow down the H/D exchange. These sample aliquots were digested for 5 min at 0°C after the addition of 1 l of a precooled pepsin solution (1 mg/ml in 5% (v/v) formic acid) and were injected directly to the mass spectrometer using a flow of 80 l/min. The MS experiments were performed with an electrospray ionization triple quadrupole instrument, model Quatro II (Micromass UK), using the same procedures described by Figueira et al. (17). The spectral data were acquired and monitored using the MassLynx software (Micromass); the spectra deconvolution of the intact protein samples was performed with the program Transform (Waters). The theoretical digest was performed using the MS-Digest web server, and the error at each data point was determined to be 0.3 Da (based on multiple measurements).
Molecular Modeling of E. coli SelA Decamer-The structural model of E. coli SelA decamer was obtained using the I-TASSER server (18) that joins multiple threading alignments to rounds of iterative structural assembly simulations for protein structure modeling.
FTIR Spectroscopy-Infrared spectra of protein solutions were collected in a Nicolet Nexus 670 FTIR spectrometer equipped with a DTGS KBr detector, corresponding to 512 scans at a resolution of 2 cm Ϫ1 over the wavenumber range 4000 -400 cm Ϫ1 at 25°C. During data acquisition, the spectrometer was continuously purged with nitrogen. The buffer spectrum was subtracted digitally from the sample spectrum. The second derivative was used to identify the peak positions of the major components of the amide I band on the original (nonsmoothed) protein vibrational spectra. To estimate the secondary structure content, Gaussian curve fitting was performed in the region of 1500 -1700 cm Ϫ1 using GRAMS/386 software package (Galactic Industries). For FTIR analyses, SelA and SPS were prepared isolated in solution but also in the combinations 1SPS:1SelA, 1SelA:1tRNA Sec , and 1SelA:1tRNA Sec :1SPS (molar ratios in monomer units). Difference infrared spectra were used to monitor the initial and the final state of SelA after SelA-tRNA Sec complex formation obtained by spectrum subtraction of the complex with the isolated samples. The final state of SPS after SelA-tRNA Sec -SPS complex formation was assessed by subtracting the experimental FTIR signal for the ternary complex, previously subtracted by the FTIR signal of the binary complex, to the SPS spectrum. The combination 1SelA:1SPS was also analyzed.
Atomic Force Microscopy (AFM)-To analyze the external dimensions, 1 l of each sample, at 0.5 mg/ml, was incubated in binding buffer without PLP for 40 min at 25°C, deposited on a mica square (10 ϫ 10 mm), and dried at room temperature for 3 h. This mica square was fixed in a metal base and analyzed in a Bruker Digital Instruments Nanoscope IIIA atomic force microscope (LNNano, CNPEM) using the non-contact mode and silicon tip of 1-nm diameter with 256 lines of scanning (19). The n-Surf 1.0 beta software was used to analyze the images and determine the dimensions of the ternary complex.

SPS Interacts with SelA-tRNA Sec Binary
Complex-To test the hypothesis that SelA-tRNA Sec interacts with SPS, we isothermally titrated 500 nM SelA-tRNA Sec binary complex fluorescein labeled with increasing amounts of dimeric SPS in the absence of their substrates. Fluorescence anisotropy of labeled tRNA Sec , covalently bound to SelA, progressively increased as a function of free SPS concentration (Fig. 1A), resulting in a specific sigmoidal binding pattern. The Hill equation (Equation 1) fitted to the experimental data with an effective dissociation constant of 610 Ϯ 79 nM and n ϭ 2.1 Ϯ 0.4, indicating positive binding cooperativity. Such a dissociation constant value is consistent with a transient biomolecular interaction.
The binding stoichiometry of the ternary complex was determined by isothermal titration of SPS in 5000 nM SelA-tRNA Sec fluorescein-labeled complex, which is above its dissociation constant value for the interaction. A progressive increase in fluorescence anisotropy was observed as a function of SPS concentration (Fig. 1B)  The observed difference in the fluorescence anisotropy initial values shown in Fig. 1, A and B, for the binary complex was larger than would typically be expected from instrument variation. It may be related to the variation of local viscosity due to the initial binary complex sample concentration being 10 times higher for the stoichiometry measurement experiment when compared with the binding measurement experiment.
H/DEx-MS Reveals the Binding Interfaces, and Fluorescence Anisotropy Spectroscopy Indicates tRNA Sec Contact Regions-H/DEx-MS followed by peptide mapping allowed the specific identification of solvent-accessible exchange sites in the dimeric SPS, the homodecameric SelA, the SelA-tRNA Sec binary complex, and the SelA-tRNA Sec -SPS ternary complex. Because SPS binding to SelA-tRNA Sec disturbs secondary structure elements of both proteins of the binary complex, altering the solvent accessibility of the contact regions, binding interfaces could be mapped by comparing the rates of H/D exchange on proteins in the bound and unbound states (17,20).
Overall, 41 peptides (including those with overlapping sequences), covering 57% of the SelA primary structure, were identified by tandem MS/MS, as shown by the coverage map ( Fig. 2A). The region from Ala 14 to Arg 17 , the SelA N-terminal domain, and regions Ala 104 -Thr 117 , Asp 146 -Cys 148 , and Ile 304 -  DECEMBER 4, 2015 • VOLUME 290 • NUMBER 49

JOURNAL OF BIOLOGICAL CHEMISTRY 29181
Lys 321 , show small percentages of deuterium incorporation, even after 30 min of deuterium exposure. Thus, these amino acid residues were hidden within the protein structure, as surface contacts in E. coli SelA decamer in solution, as observed in the crystallographic structure of the homologous A. aeolicus SelA (4).
Following SelA-tRNA Sec covalent binding, we detected 41 peptides (including those with overlapping sequences), covering 63% of the SelA amino acid sequence ( Fig. 2A). Characterization of the solvent accessibility of the N-terminal domain shows that regions Leu 27 -Gly 31 and Leu 40 -Ile 51 are hidden after SelA-tRNA Sec binding (Fig. 2B). These regions were recently observed to interact with tRNA Sec D-loop in the crystallographic structure of A. aeolicus SelA-tRNA Sec (4). Other regions, including fragment Leu 137 -Ala 154 and the amino acid residues near the active site (Lys 295 ), also have low incorporation of deuterium even after 30 min of exposure (Fig. 2, B and C and supplemental Tables 1A and 1B). These regions must be non-covalent SelA-tRNA Sec contacts on the surface of SelA.
In addition, evaluation of the effect of stereo chemical block in tRNA Sec interaction with SelA by qualitative fluorescence anisotropy spectroscopy assays showed a decrease in SelA-tRNA Sec observed binding when the acceptor arm, D-loop, and variable arm were mutated for the corresponding E. coli tRNA Ser region, highlighting the importance of these regions in SelA-tRNA Sec specific interaction (Fig. 3, A-G). As a negative control, we titrated fluorescein-labeled single-stranded DNA (Fig. 3G). The interaction pattern of SelA-(mutant) tRNA Sec binding is similar to that previously observed by Manzine et al. (6) and does not present a saturation plateau because decameric SelA can stack side-by-side and one on top of each other.
The anticodon and T⌿C arms variations did not affect the SelA-tRNA Sec interaction (Fig. 3, A and B, respectively); however, the substitution of the D-loop by a fragment from E. coli tRNA Ser D-loop caused a decrease in the binary complex interaction (Fig. 3C). These results highlight the D-loop as responsible for the specificity of SelA-tRNA Sec recognition, which corroborates with the SelA-tRNA Sec binary complex crystallographic structure from A. aeolicus (Protein Data Bank (PDB) ID 3W1K (4)). Based on amino acid sequence alignment between E. coli and A. aeolicus SelA (data not shown) and A. aeolicus SelA-tRNA Sec structure analysis (4), we identified by H/D-Ex MS the E. coli SelA Leu 27 -Gly 31 and Leu 40 -Ile 51 regions as interaction points to E. coli tRNA Sec D-loop and T⌿C arms (Fig. 2, A and B).
The deletion of the variable arm or its substitution by the E. coli tRNA Ser variable arm (Fig. 3, D and E, respectively) and the acceptor arm reduction from 8ϩ5 to 7ϩ5 (Fig. 3F) caused a marked decrease in the anisotropy values. The 8ϩ5 folding is a key difference to other tRNAs and must be an important SelA recognition point that was not identified based on structural analysis (4).

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Mapping the surface interactions of SelA to form the ternary complex shows that the N-terminal region (Glu 46 -Arg 52 ) of SelA and two small loops (Glu 67 -Asp 69 and Ala 111 -Thr 117 ) have low deuterium incorporation when compared with SelA in the binary complex (Fig. 2B). We believe that these are the most important SelA-SPS interaction regions.
For SPS, 41 peptides were identified, covering 68.7% of the primary structure. Amino acid residues Leu 136 -Asp 143 , Ser 239 -Gly 245 , and Pro 271 -His 283 presented low rates of deuterium incorporation even after 30 min of exposure. These regions are hidden within the protein and either are near or participate in the SPS dimerization interface (Fig. 2, B and D). The SPS N-terminal loop showed a high deuterium incorporation rate after 5 min of exposure, indicating that it is a flexible region.
It is worth noting that within 30 min, 68.7% of the amide hydrogen atoms in SPS were replaced with deuterium, whereas only 62.5% were replaced in the presence of the SelA-tRNA Sec binary complex, indicating that some amide protons were protected from deuterium exchange upon ternary complex forma-tion. The SPS N-terminal flexible loop (Met 1 -Thr 9 ) is hidden from H/D exchange after the interaction of SPS with the binary complex, resulting in lower deuterium incorporation. Two other loop regions (Leu 43 -Val 54 and Met 71 -Pro 72 ) and an ␣-helix region (Glu 120 -Cys 129 ) that are near the catalytic site of the dimeric enzyme become inaccessible to the solvent after the interaction (Fig. 2, B and 2D). Our data identify the regions of molecular contact between the various components of the ternary complex and indicate that the regions near the active sites are crucial to the interaction between SPS and SelA-tRNA Sec to form a ternary complex.
FTIR Spectroscopy Suggests SPS Conformational Changes-Structural changes due to SPS binding to the SelA-tRNA Sec binary complex were investigated via FTIR spectroscopy because the amide I region (1600 -1700 cm Ϫ1 ) of the FTIR spectra is sensitive to changes in the protein secondary structure (21)(22)(23)(24). SPS and SelA amide I bands were resolved into seven bands each. The bands appearing at 1628 and 1676 cm Ϫ1 are attributed to the low-and high-frequency components of (1) 1621.7 cm Ϫ1 . C, comparison between the dimeric SPS infrared spectrum and the spectrum observed after ternary complex formation. D, infrared spectrum difference between dimeric SPS and SPS bound to the SelA-tRNA Sec complex previously subtracted by the SelA-tRNA Sec FTIR signal.
We observed that the amide I absorption band of SelA did not change upon SelA-tRNA Sec interaction when we analyzed the difference spectrum between SelA-tRNA Sec binary complex and SelA, which implies that SelA does not have a significant secondary structure variation upon tRNA Sec binding. Additionally, concerning the ternary complex formation, we propose that the most significant secondary structure change is more likely to be in SPS.
Indeed, there is an evident shift in the amide I absorption band of SPS (Fig. 4C) upon its binding to the SelA-tRNA Sec binary complex when compared with the SPS sample, indicating that SPS undergoes a conformational change to form the ternary complex. Such a shift was not observed in the absence of tRNA Sec , implying that the SelA-SPS interaction is dependent on previous tRNA Sec interaction with SelA.
To further analyze the change in the secondary structure of SPS after its interaction with the SelA-tRNA Sec binary complex, we obtained a difference spectrum by subtracting the spectrum of free SPS from that of the bound protein, which was previously subtracted by the contribution of SelA-tRNA Sec (Fig. 4D). The result shows a large negative band of ϳ1653 cm Ϫ1 and a positive band in the 1640 -1620-cm Ϫ1 range. This pattern can be due to the loss of an ␣-helical component, as first described by Trewhella et al. (24), indicating that a structural element in the ␣-helix configuration in SPS loses conformation to enable the formation of the ternary complex SelA-tRNA Sec -SPS.

Functional Assay Reveals That Selenoprotein Synthesis in E. coli Is Dependent on N-terminal Regions of SPS and SelA-
Because the H/D change experiment strongly suggested the participation of the SPS N-terminal loop in the SelA-tRNA Sec -SPS complex assembly, we investigated the potential role of this region in selenoprotein biosynthesis. Previous in situ limited proteolysis experiments with chymotrypsin protease removed the first 11 residues of E. coli SPS (⌬11-SPS) (data not shown), but the catalytic residues Cys 17 and Lys 20 were preserved. Fluorescence anisotropy of SelA-tRNA Sec is not altered with ⌬11-SPS titration, indicating a lack of specific interaction between ⌬11-SPS and the binary complex (Fig. 5A). Functional complementation assays in E. coli strain WL400, which lacks the SPS gene, transformed with ⌬11-SPS, were unable to restore the selenoprotein biosynthesis (Fig. 5B), despite the presence of the known catalytic residues. The positive control WL400 transformed with the E. coli SPS gene (Fig. 5C) developed the purple color characteristic of selenoprotein biosynthesis. We also investigated whether this region is required for assembly of the SelA-tRNA Sec -SPS complex. SPS multiple sequence alignment analysis revealed three highly conserved residues (Leu 8 , Thr 9 , and Tyr 11 ) in the SPS N-terminal sequence; however, the biological significance of these residues has not yet been investigated. Together, these results suggest that the SPS N-terminal region is essential to SelA-tRNA Sec -SPS complex assembly and that its deletion impairs selenoprotein biosynthesis.
Additionally, because H/D exchange experiments showed that the N-terminal domain of SelA is part of its decamerization interface, we also tested its requirement in Sec synthesis in a functional complementation assay in the E. coli strain JS1, which lacks the selA gene. N-terminally truncated SelA was unable to restore Sec synthesis (Fig. 5D) as seen in the positive control E. coli SelA (Fig. 5E). It is worth noting that Methanocaldococcus jannaschii SelA, which lacks an equivalent N-terminal domain but shares 30% amino acid sequence identity with E. coli SelA, is organized as a non-functional dimer and does not interact with tRNA Sec (25).
SelA-tRNA Sec Binary Complex Dimensions Are Compatible with SPS Interaction-Engelhardt et al. (26) were the first to visualize, in 1992, the decamers of SelA and SelA-tRNA Sec by transmission electron microscopy of negative stained samples. Manzine et al. (6) determined the stoichiometry of the binary complex (SelA-tRNA Sec ) as 1SelA decamer :10tRNA Sec . This stoi- chiometric ratio, different from the accepted 1SelA decamer : 5tRNA Sec , was fundamental for investigating the conformational changes occurring in the transition from a binary to a ternary complex. We used AFM to measure the low-resolution dimensions of SelA decamer as 20.8 Ϯ 0.5 nm in diameter and 3.96 Ϯ 0.05 nm in height as the average for 58 single particles. After the binding of 10 tRNA Sec , the average dimensions of the binary complex were 22.0 Ϯ 0.5 nm in diameter and 3.56 Ϯ 0.05 nm in height from 86 single particles. The decrease in height is consistent with the size of the predicted SPS interaction surface (Fig. 6A) allowing the SelA-tRNA Sec -SPS interaction. The lowresolution dimensions of the SelA-tRNA Sec -SPS ternary complex were 27.0 Ϯ 0.5 nm in diameter and 4.02 Ϯ 0.05 nm in height, as determined from the average of 58 single particles.

Discussion
Sec biosynthesis in E. coli requires 10 molecules of Ser-tRNA Sec covalently bound to homodecameric SelA to catalyze the conversion of Ser to Sec (5). The SelA-tRNA Sec binary complex can thus be interpreted as a reservoir of cellular tRNA Sec .
It was observed by H/DEx-MS presented here that the N-terminal region of SelA is required for SelA oligomerization, as it becomes hidden from the surface of homodecamers exposed to solvent. Therefore, homodecamerization, and consequently, the Ser-Sec conversion and selenoprotein biosynthesis, is dependent on the N-terminal region (or N terminus), as we observed by functional complementation with the N-terminally truncated E. coli SelA. Similar results from A. aeolicus SelA N-terminal mutants (27) and the non-functional dimeric M. jannaschii SelA (25), which do not interact with tRNA Sec , strengthen our findings.
A Schiff base is formed between the ␣-amino group of the Ser residue with the formyl group of PLP following SelA-tRNA Sec interaction, resulting in the synthesis of the intermediate aminoacrylyl-tRNA Sec upon dehydration of the amino acid residue (5). FTIR experiments show that SelA does not undergo a secondary structure change upon its interaction with tRNA Sec as was also observed in the crystallographic structure of A. aeolicus SelA-tRNA Sec complex (4), and fluorescence anisotropy spectroscopy with tRNA Sec mutants has shown that this interaction is dependent on the tRNA Sec acceptor arm, D-loop, and variable arm.
In addition to the D-loop arm (4) as the recognition point of tRNA Sec to SelA, we observed that the difference in the acceptor arm pairing number (8 to 7) is essential for tRNA Sec affinity to E. coli SelA. Selenium is transferred to the aminoacrylyl-tRNA Sec intermediate complex in the form of selenophosphate, a product of dimeric SPS selenide water dikinase catalytic activity (7,10), to form Sec-tRNA Sec . The SPS dimerization interface is composed of the ␤-sheet domain of each monomer, a common structural characteristic of the PurM protein superfamily (7). This dimerization domain was confirmed by our H/DEx-MS experiments. In addition, consistent with the SPS crystallographic structures (PDB ID 3U0O) that were previously described, the glycine-rich N-terminal region of SPS was observed to be flexible in solution, showing high levels of deuterium exchange even after low deuterium exposure time. This flexibility allows the formation of the SPS active site on its "closed" form, upon ATP binding, releasing the catalysis product in its "open" form (7,10).
Because one SelA decamer molecule and 10 tRNA Sec molecules form a covalently bound binary complex (6), we analyzed the interaction of SPS with the SelA-tRNA Sec complex. Using fluoresceinlabeled tRNA Sec , we observed an increase in fluorescence anisotropy following SPS isothermal titration, revealing a specific binding leading to the formation of the ternary complex, with a stoichiometric ratio of one SelA decamer covalently bound to 10 tRNA Sec molecules interacting with five SPS dimers. The SelA-tRNA Sec -SPS interaction dissociation constant of 610 Ϯ 79 nM is consistent with the expected values for biomolecular transient interactions. Hill's plot (Fig. 1A) indicates a positive cooperativity, with n ϭ 2.1 Ϯ 0.4, for the formation of the ternary complex. Based on this observation, we propose that trapping the selenium compounds in the SelA-tRNA Sec -SPS complex would be an efficient mechanism to avoid the high cellular toxicity posed by free selenium. Additional experiments are necessary to verify this hypothesis.
The SelA-tRNA Sec -SPS interaction is dependent on stereochemical recognition, involving the structural accommodation of one molecule to the other. Remarkably, the height of the FIGURE 6. Dimension analysis of SelA, SelA-tRNA Sec , and SelA-tRNA Sec -SPS complex by AFM. The samples were analyzed using low concentrations, 0.5 mg/ml, dried in mica grids. A, SelA; B, SelA-tRNA Sec ; C, SelA-tRNA Sec -SPS. Grids were observed using a NanoScope III atomic force microscope (Digital Instruments) and analyzed using n-Surf 1.0 beta software (n-Surf).