Dimer-dimer interaction of the bacterial selenocysteine synthase SelA promotes functional active site formation and catalytic specificity

Abstract

The 21^st amino acid, selenocysteine (Sec), is incorporated translationally into proteins, and is synthesized on its specific tRNA (tRNA^Sec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA^Sec, formed by seryl-tRNA synthetase, to Sec-tRNA^Sec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate (PLP)-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA^Sec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA^Sec. The SelA catalytic site is close to the dimer-dimer interface, but the significance of the dimer-pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions, and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer-dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site, and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I PLP-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.

Introduction

Selenocysteine (Sec) is known as the 21st amino acid incorporated translationally into proteins, and is synthesized on its specific tRNA (tRNA^Sec).¹ The tRNA^Sec-ligated serine (Ser-tRNA^Sec) generated by seryl-tRNA synthetase (SerRS) is converted to tRNA^Sec-ligated Sec (Sec-tRNA^Sec), where selenophosphate, synthesized by selenophosphate synthetase (SPS), is used as a selenium donor.² In Eukaryotes and Archaea, Ser-tRNA^Sec is phosphorylated by O-phosphoseryl-tRNA^Sec kinase (PSTK) to generate O-phosphoseryl-tRNA^Sec (Sep-tRNA^Sec),³ followed by Sep to Sec conversion catalyzed by SepSecS.^{4; 5} On the other hand, in Bacteria the selenocysteine synthase SelA directly converts Ser-tRNA^Sec to Sec-tRNA^Sec, without a previous phosphorylation step.⁶ Both SelA and SepSecS are pyridoxal 5′-phosphate (PLP)-dependent enzymes.

SelA is a homodecameric enzyme with an overall molecular mass of about 500 kDa. We previously determined the crystal structures of SelA with and without tRNA^Sec.⁷ The SelA decamer is organized as a pentamer of intimate dimers, and it binds ten tRNA^Sec molecules. Residues from each of the four SelA subunits of two intimate dimers are involved in processing one Ser-tRNA^Sec molecule, indicating that the decameric quaternary structure is essential to bind Ser-tRNA^Sec.⁷ We proposed the SelA catalytic mechanism based on the co-crystal structure with thiosulfate, an analog of selenophosphate. While the tRNA binding pocket is formed at the dimer-dimer interface, the significance of the formation of an (α₂)₅ SelA structure in the generation of functional catalytic sites remained unclear.

The enzymes SelA and SepSecS belong to the largest PLP-dependent enzyme group, the fold-type-I superfamily, and catalyze the replacement of the amino acid β position (β replacement), in an α,β elimination reaction proceeding via a 2-aminoacrylate intermediate, and a subsequent addition step (Fig. 1).⁶ Although β replacement is a common reaction, there are two major difficulties with the Sec synthesis step. First, the precursor, Ser-tRNA^Sec, lacks the α-carboxyl group, which is used for the substrate-enzyme interaction in all fold-type-I PLP-dependent enzymes that work with α-amino acids.⁸ Second, the leaving group in the β-elimination step is a hydroxide ion from the Ser moiety (Fig. 1).⁶ In general, a hydroxide ion is not a suitable leaving group, and no other fold-type-I members can eliminate it from the β-position of an α-amino acid. In Eukarya/Archaea, these difficulties are overcome with the assistance of PSTK. PSTK phosphorylates the Ser moiety to generate Sep-tRNA^Sec,³ which is the substrate of SepSecS (Fig. 1). The β elimination is thus facilitated, because the phosphate is an excellent leaving group. Furthermore, the phosphate group is predominantly responsible for the substrate binding to SepSecS.⁹ In contrast, the bacterial system differs strikingly, in that the single enzyme SelA directly converts Ser-tRNA^Sec to Sec-tRNA^Sec, without phosphorylation. Our previous study suggested a putative binding site for the 3′-terminal A76 of tRNA^Sec, and the binding site could compensate for the lack of the α-carboxyl group.⁷ However, the mechanism that facilitates the hydroxyl-group elimination has not been elucidated.

Fig. 1. The enzymatic reaction.

Schematic views of the enzymatic reactions of SelA, SepSecS, and cystathionine β-lyase (CBL). These enzymes catalyze α-position deprotonation, followed by β-position elimination, to generate tRNA-ligated or non-esterified 2-aminoacrylate intermediates, conjugated with PLP. The substrate of CBL is cystathionine, and the enzyme-substrate interaction is responsible for the α-carboxyl group,³¹ (c), while SepSecS interacts with the phosphate group of its substrate Sep-tRNA^Sec.⁹ However, Ser-tRNA^Sec, the substrate of SelA, has neither a carboxyl nor a phosphate group. Furthermore, the leaving group in the SelA-catalyzed reaction is a hydroxide ion, which is less suitable as compared to the phosphate and the deprotonated homocysteine eliminated in the SepSecS- and CBL-catalyzed reactions, respectively.

In this study, we have determined the structural basis for productive SelA active site formation, which requires the cyclization of SelA dimers to a full decamer. Analyses of the structures of Aquifex aeolicus SelA and dimeric SelA variants revealed that the active site conformations of SelA intimate dimers differ significantly from those of decameric SelA. Structural rearrangements upon decamerization generate ten catalytically functional active sites that properly accommodate and position Ser-tRNA^Sec for Sec formation. Based on a comparison of SelA with phylogenetically related enzymes, we describe a refined mechanism for the direct hydroxyl-group elimination facilitated by SelA, and propose an evolutionary hypothesis for the emergence of the decameric SelA architecture.

Results and discussion

The dimer-dimer interface in the pentamer-of-dimers structure of SelA

A. aeolicus SelA is a homodecamer, in which the ten subunits, designated here as subunits A to J, form a “pentamer of dimers” (Fig. 2a). The “intimate dimers” A•B to I•J have the largest intersubunit interfaces (e.g., A•B interface, 4,067 Ų), on which the enzyme active sites are built. Therefore, the intimate dimer is the basal building block of SelA. In the pentamer-of-dimers quaternary structure, intimate dimers I•J and A•B, for example, interact with each other through subunits J and A. The dimer-dimer interface encompasses 1,140 Ų, and characteristically constitutes the overall decameric arrangement of SelA. The dimer-dimer interface may be divided into three parts. The first part (part I, 438 Ų) has internal two-fold symmetry, and is composed of two sets of residues from Asn218 to Phe224, constituting strand β11 within the core domain (Fig. 2b). Strand β11 from subunit J, for example, and the corresponding strand from subunit A form an antiparallel β-sheet between intimate dimers I•J and A•B. The other parts, II and III (307 Ų each), are symmetrically related to each other. Parts II/III between subunits J and A comprise Arg163, Leu166, Glu168, and Arg174 (η4-loop-β6-loop-β7) from subunit J, and Glu188, Thr191, Thr192, Asn193, Lys196, and Asp199 (β8-loop-β9-α8) from subunit A, and vice versa (Fig. 2c).

Fig. 2. Structure of SelA.

(a) Overall view of the crystal structure of decameric SelA-FL. PLPs are represented as sphere models. The N-terminal domain, the N-linker, the core domain, and the C-terminal domain of subunit A are colored cornflower blue, magenta, blue, and medium-purple, respectively, while those of subunit B are colored green-yellow, yellow, green, and aquamarine, respectively. Subunits C–J are colored gray.

(b, c) Close-up views of the interactions at interfaces I (b) and II (c) between intimate dimers. Interactions between subunits A and J are shown. The colors of the ribbon diagrams are the same as in (a).

(d, e) Overall views of the dimeric SelA mutants, dSelA-FL (d) and dSelA-ΔN (e). The ribbon diagrams are colored as in panel a. dSelA-FL lacks PLP.

We introduced mutations into the dimer-dimer interface, by using Escherichia coli SelA (Table 1). The amino acid residues in the dimer-dimer interface are conserved between the A. aeolicus and E. coli SelAs, although the conservation in part I is relatively low (Table 1). The Mut1 and Mut2 mutations were designed to impair the antiparallel β11•β11 interaction in part I, by deleting residues 222–225 and 222–223, respectively (residue numbering according to A. aeolicus SelA). The enzymes bearing the Mut1 and Mut2 mutations were catalytically inactive in vivo (Table 1), indicating that the integrity of strand β11 and the formation of the resultant antiparallel β-sheet are critical for the function of SelA. Mutations targeting parts II/III were also examined (Table 1). Here, the Ala mutations of individual residues in parts II/III exhibited no significant effects on the SelA activity (Mut3, 6, and 7a). In contrast, the Thr191Tyr, Thr192Tyr, and Asp199Arg mutations (Mut4, 5, and 7b), which were designed to cause steric clashes, inactivated E. coli SelA in vivo. Similarly, the deletion of Thr192–Asn193 (Mut8), or the replacement of four residues, Thr191–Lys194, with two Ala residues (Mut9) rendered E. coli SelA inactive in vivo. These results suggested that the integrity of parts II/III is also important for the SelA activity.

Table 1.

In vivo mutational analyses of E. coli SelA

SelA mutant	Amino acid change E. coli numbering	A. aeolicus Numbering	Function of residue	PLP occupancy (Mut10a–Mut10d) (%)	Activity in vivo
1	ΔGln233–Thr236	Glu222–Val225	Decamerization interface I		−
2	ΔGln233–Gly234	Glu222, Gly223	Decamerization interface I		−
3	Arg185Ala	Arg174	Decamerization interface II/III		+++
4	Thr202Tyr	Thr191	Decamerization interface II/III		−
5	Thr203Tyr	Thr192	Decamerization interface II/III		−
6	His207Ala	Lys196	Decamerization interface II/III		+++
7a	Asp210Ala	Asp199	Decamerization interface II/III		+++
7b	Asp210Arg	Asp199	Decamerization interface II/III		−
8	ΔThr203–Asn204	Thr192, Asn193	Decamerization interface II/III		−
9	Thr202–Arg205 = 2 × Ala	Thr191–Lys194	Decamerization interface II/III		−
10a	Lys295Ala	Lys285	Covalent PLP ligand	n.d.	−
10b	Lys295Ala, Asp294Lys	Lys285, Asp284	Covalent PLP ligand	25	−
10c	Lys295Ala, Leu296Lys	Lys285, Leu286	Covalent PLP ligand	37	−
10d	Lys295Ala, Leu297Lys	Lys285, Leu287	covalent PLP ligand	30	−

+++, fully active; +, significantly reduced activity; −, inactive; n.d., below detection limit.

Mutations introduced in E. coli SelA, numbering of the corresponding residues for A. aeolicus SelA, and functions of the residues as determined from the structures and catalytic activities of the corresponding E. coli SelA variants in vivo are indicated. PLP occupancy for SelA variants 10a–10d is indicated. All SelA variants lacking catalytic activity in vivo were purified and tested for solubility to exclude the possibility that the activity loss was due to inclusion body formation.

Crystal structure of dimeric SelA

The interface between the intimate dimers is much smaller than that between the subunits within the intimate dimer, suggesting weaker interactions between the dimer units. In fact, a quadruple mutation (Tyr220Pro-Asp199Arg-Thr191Tyr-Thr192Tyr) abolished the pentamerization, and resulted in an inactive “dimerized” or “depentamerized” enzyme.⁷ (In the following, we refer to the depentamerized enzyme as dimeric SelA). Importantly, dimeric SelA is stable and can be purified for crystallization. In this study, we crystallized the full-length and N-terminally-truncated (ΔN, deletion of residues 1–61) dimeric SelA proteins, and determined their structures at 3.35 and 2.40 Å resolutions, respectively (Fig. 2d and e). The lysine residues of the dimeric full-length SelA (dSelA-FL) were methylated for crystallization improvement, while the dimeric SelA-ΔN (dSelA-ΔN) was crystallized without Lys methylation. The overall structures of dSelA-FL and dSelA-ΔN are similar to each other, and to those of the intimate dimers of decameric SelA-FL and SelA-ΔN, respectively. Each subunit of dSelA-FL consists of the N-terminal domain (residues 1–66), the N-linker (residues 67–89), the core domain (residues 90–338), and the C-terminal domain (residues 339–452) (Fig. 2d). The β11 strand in part I of the dimer-dimer interface is disordered in the dSelAs.

Catalytic site of dimeric SelA

The catalytic site is located at the subunit interface, involving the core domains and the N-linker, within the intimate dimer unit of SelA. PLP is covalently bound to Lys285, as a Schiff base. Surprisingly, the catalytic-site structures of dSelA-FL and dSelA-ΔN differ distinctly from that of the SelA decamer (Fig. 3a to c). The dimeric SelAs lack a SelA-specific β sheet (β6•β7), and α7 has a distinct orientation. These different catalytic-site conformations of the dimeric mutants resemble those of other fold-type-I PLP-enzymes (Fig. 3b, c, e to h). The position of PLP in the dSelA-ΔN catalytic site is the same as that in the SelA decamer, while dSelA-FL lacked PLP in its catalytic site. PLP may have been removed during the Lys methylation of dSelA-FL, whereas the decameric SelA-FL and SelA-ΔN retained PLP during the methylation.⁷ This indicated that the depentamerization increased the accessibility of Lys285, which probably resulted in the replacement of PLP by a methyl group, although the methyl group was not visible in the structure due to the high mobility of the Lys285 side chain.

Fig. 3. Catalytic site comparison.

(a, b, c) Catalytic site comparison between the decameric and dimeric SelAs. The β sheet β6•β7 and α7 in the catalytic site also participate in the pentamerizing interaction in the decameric SelA-ΔN (a). On the other hand, dSelA-FL (b) and dSelA-ΔN (c) lack the β6•β7 sheet, and α7 changes its orientation; therefore, the catalytic-site structure is disrupted. dSelA-FL also lacks PLP. Thiosulfate ions are bound in different sites among (a) to (c). Possible hydrogen bonds or ionic interactions are shown as dashed lines.

(d) Close-up view of the TS4 and TS5 binding sites of dSelA-ΔN. The 2F_O–F_C electron density map at the 1.0 σ level is shown.

(e, f, g, h) Catalytic sites of SepSecS (PDB ID: 2Z67³²) (e), SepCysS (PDB ID: 2E7J³³) (f), CBL (PDB ID: 1CL1³³) (g), and MJ0158 (PDB ID: 2AEV¹⁶) (h). They lack the β6•β7 counterpart, and the orientations of their α7 counterparts are closer to those of α7 in depentamerized SelA mutants. The phosphate/sulfate ions at the catalytic site are shown. The SepSecS structure 2Z67 is from the archaeon Methanococcus maripaludis. The focused Arg and Lys residues are labeled according to their numbering in human/M. marpaludis SepSecS.

The thiosulfate-binding sites also differ from those of the decameric SelA-ΔN (Fig. 3a to c). The decameric SelA-ΔN binds three thiosulfate ions (TS1–TS3) in each catalytic site. TS1 may mimic the substrate selenophosphate, and TS2 and TS3 may mimic the phosphate groups of the nucleotides A76 and C75, of the tRNA^Sec CCA terminus.⁷ In contrast, dSelA-ΔN binds two thiosulfate ions, TS4 and TS5. TS4 may correspond to TS1, but TS5 does not correspond to any of TS1–TS3. TS1 interacts with Arg86^A (subunit A residue), and Arg312^B and Arg315^B (subunit B residues), whereas TS4 interacts with Ser172^A, Arg174^A, Arg116^B, and Arg315^B (Fig. 3d). The distance between TS1 and TS4 is about 3.5 Å. TS5 interacts with Arg312^B and Arg315^B. dSelA-FL also has two thiosulfate ions, TS5 and TS6. TS5 is the same as that in dSelA-ΔN, whereas TS6 occupies the position of the PLP phosphate group. The difference between the thiosulfate-binding sites of dSelA-ΔN and dSelA-FL may be caused by the lack of PLP in the catalytic sites of dSelA-FL.

In decameric SelA, the β6•β7 sheet is stabilized by the pentamerizing interactions. The main chain NH and carbonyl moieties of Glu168^A in β6 interact with Asn193^J, and the Arg174^A side chain forms a salt bridge with Asp199^J (Fig. 3a). Since Arg174^A also participates in α7, the α7 orientation is dependent on the Arg174^A position, which is fixed by the Arg174^A•Asp199^J salt bridge. Although the β6•β7 sheet has no polar interactions with the thiosulfate ions, Gly170^A in the β6–β7 loop is close to TS1 and forms a van der Waals interaction (Fig. 3a). Therefore, the β6•β7 sheet contributes to TS1 binding and is important for catalysis.

These results indicated that the formation of the catalytic site structure is dependent on the pentamerization of intimate dimers, and that the dimeric mutant cannot properly bind its substrates. The activity loss by the mutations introduced to the pentamerization interfaces (Table 1) should result from both the prevention of decamer-dependent tRNA^Sec binding and the disruption of the catalytic-core structure.

How does SelA eliminate the hydroxyl group from the Ser moiety?

In general, proper binding and hydroxyl-group protonation of the substrate are essential for an enzyme to eliminate a hydroxyl group. SelA has a putative binding pocket for the A76 adenine ring (Fig. 4a),⁷ and can therefore accommodate the Ser moiety of Ser-tRNA^Sec. This binding pocket is critically important, because Ser-tRNA^Sec lacks the α-carboxyl group, which is used for the substrate-enzyme interaction in all of the fold-type-I superfamily PLP-dependent enzymes working on α-amino acids.⁸

Fig. 4. Proposed scheme of the SelA-catalyzed reaction.

(a) Docking model of the Ser-ligated terminal nucleotide (A76) of tRNA^Sec and Se-P. A76 and the Ser moiety are modeled based on the mutational analyses, and their carbon atoms are colored salmon and spring green, respectively. Arg86 is located in the vicinity of Lys285 and Ser. Phe224 from subunit J and Asn218 from subunit A form the A76-binding pocket. The Ser moiety is attached covalently to PLP. Selenophosphate (Se-P) occupies the TS1 binding site.

(b) The reaction scheme of the protonation of the β hydroxyl group by Lys285, which enables the elimination of the leaving group (a water molecule). Arg86 may provide the basic environment to strengthen the Lys285 protonation activity.

(c) The interaction interface between the core and C-terminal domains of SelA. The N-linker (magenta) and the SelA-specific insertions I and IV (cyan) participate in the interaction between the core and C-terminal domains. The other regions are colored as in Fig. 2a. β1 in the N-linker forms a β sheet with β18 in the C-terminal domain. Arg86 in the N-linker participates in the catalytic site.

To examine the mechanism of hydroxyl-group protonation, we compared SelA with other PLP-dependent enzymes. SelA is the only fold-type-I enzyme that eliminates the hydroxyl group from the substrate amino acid (or amino acid moiety). Therefore, we compared SelA with cystathionine β synthase (CBS), a fold-type-II PLP-dependent enzyme that catalyzes a β-replacement reaction. CBS eliminates the hydroxyl group from serine to generate 2-aminoacrylate, and then adds homocysteine to synthesize cystathionine (Supplementary Fig. 1a). The β elimination step is the same as that in SelA catalysis (Fig. 1). A crystallographic study revealed that Tyr227 of CBS is responsible for the protonation of the serine hydroxyl group.¹⁰ Interestingly, cysteine synthase (CysS), the close homolog of CBS, lacks the corresponding Tyr residue.¹¹ CysS catalyzes the β replacement reaction that eliminates an acetyl group from O-acetylserine, and adds hydrogen sulfide to generate cysteine (Supplementary Fig. 1b). In this reaction, the leaving group is acetate, and thus hydroxyl-group protonation is unnecessary.

On the other hand, SelA lacks residues such as Tyr, His, and Cys that could serve as proton donors in the vicinity of the Ser-moiety binding site, except for Lys285, which forms a Schiff base with PLP in the waiting state. Some enzymes in the sugar aminotransferase subfamily catalyze the elimination of the α-hydrogen and the β-hydroxyl group. Here, the α position indicates the position of the amino group conjugated with PLP during catalysis. The enzymes GDP-6-deoxy-α-D-lyxo-hexopyranos-4-ulose dehydratase (ColD) and CDP-6-deoxy-α-D-xylo-hexopyranos-4-ulose dehydratase (E₁) employ a His residue, which is conserved at the position of the PLP-coordinating Lys285 of SelA, to catalyze the α,β elimination step (Supplementary Fig. 1c and d).^{12; 13} In contrast, 5-amino-3-hydroxybenzoate (AHBA) synthase, another enzyme that eliminates the α-hydrogen and the β-hydroxyl group (Supplementary Fig. 1e), contains a conserved Lys residue, which is considered to protonate the β-hydroxyl group.¹⁴ Here, the generation of a benzene ring in the product is likely to promote the α,β elimination reaction.

These findings suggested that Lys285 in SelA can act as proton donor for the β-hydroxyl group (Fig. 4b). As enzymes like ColD and E₁ utilize His, instead of Lys, to strengthen the protonation activity, and AHBA generates a benzene ring to promote the protonation and elimination of the β-hydroxyl group, an additional auxiliary mechanism to support protonation and β-hydroxyl elimination might be plausible for SelA as well. Such a mechanism would involve the Arg86 residue (Fig. 4a and b). Our in vivo mutational assay revealed that Arg86 is mandatory for activity.⁷ Arg86 is located in the vicinity of Lys285 (Fig. 4a), and the positive charge of Arg86 may reinforce the protonation activity of Lys285. Arg86 is located in the N-linker between the N and core domains. The β strand β1 in the N-linker forms a β sheet with β18 from the C-terminal domain (Fig. 4c). Although the corresponding β sheet is present in many fold-type-I PLP-dependent enzymes, the C-terminal domain orientation in SelA differs from those of the other homologues (Fig. 5). The unique C-terminal domain orientation in SelA results in the specific positioning of the N-linker that allows participation in the catalytic site. This structural feature is the reason why the N-linker can provide Arg86 in the vicinity of Lys285. Here again, the characteristic tertiary and quaternary structures of SelA are essential for its unique catalytic activity to eliminate the hydroxyl group.

Fig. 5. Structure comparison with other fold-type-I PLP-dependent enzymes.

(a, b, c, d) Overall crystal structures of SepSecS (PDB ID: 2Z67³¹) (a), SepCysS (PDB ID: 2E7J³²) (b), cystathionine β-lyase (CBL) (PDB ID: 1CL1³³) (c), and MJ0158 (PDB ID: 2AEV¹⁶) (d). The N-terminal domains, the N-linkers, the core domains and the C-terminal domains of subunits A and B are colored as in Fig. 2a. The N-terminal regions of SepCysS and MJ0158 correspond to the SelA N-linker. Like the SelA N-linker, these regions form β sheets with their C-terminal domains. On the other hand, the N regions of the homotetrameric enzymes SepSecS and CBL participate in the interactions between the two intimate dimers, and thus they occupy different positions from that of the SelA N-linker. The intimate dimers C•D of SepSecS and CBL are colored gray in the top panels, and are not shown in the bottom panels. (e, f, g, h) Comparison of the C-terminal domain orientation with SelA. The A subunits of SepSecS (e), SepCysS (f), CBL (g), and MJ0158 (h) were superposed on the SelA subunit A, by fitting their core domains. The core and C-terminal domains of SelA are colored blue and medium-purple, respectively, as in Fig. 2a, while those of the other PLP-dependent enzymes are colored light blue and yellow, respectively. The major helices α12 and α14, located in the core and C-terminal domains, respectively, are connected by the short helices η7 and α13 in SelA. In contrast, the α12 and α14 counterparts are directly connected in SepSecS, and form a continuous single helix in SepCysS and CBL. On the other hand, MJ0158 has the α13 counterparts, and the orientation of its C-terminal domain is somewhat closer to that of the SelA C-terminal domain.

SepSecS also possesses an Arg residue (Arg75 in human SepSecS) that occupies the space corresponding to SelA Arg86.⁹ However, Arg75 is located in the core domain of SepSecS and is not an alignment-based counterpart of SelA Arg86, which is in the N-linker. In fact, Arg75 forms an inter-subunit interaction with Lys-bound PLP; i.e., Arg75^B interacts with PLP-Lys284^A (Fig. 3e). In contrast, the Arg86 and PLP-Lys285 interaction is an intra-subunit one (Figs. 3a and 4a). Moreover, the PLP-ligated Lys284 in SepSecS cannot protonate the β-hydroxyl group of the Ser moiety; SepSecS cannot bind Ser-tRNA^Sec because the phosphate group of the Sep moiety is essential for the substrate Sep-tRNA^Sec binding.

Why did two different pathways for Sec formation evolve?

Considerable conservation of the translation machinery components exists among the three domains of life. However, the pathways of aminoacyl-tRNA formation evolved much more independently. For instance, the synthesis of Asn-tRNA or Gln-tRNA involves tRNA-dependent and tRNA-independent amino acid biosynthesis routes.¹⁵ In the case of Sec, nature also designed distinct routes based on the different enzyme structures operating in Bacteria versus Archaea/Eukaryotes. Our phylogenetic analysis (Fig. 6) revealed that SelA is related to the cystathionine γ-synthase (CGS) family members catalyzing the β-elimination of cysteine derivatives, or the γ-elimination and γ-replacement of homocysteine products. Thus, the ancestor of SelA may have been an enzyme involved in sulfur metabolism. The protein closest to SelA in the phylogenetic tree is the archaeal MJ0158, a homodimeric protein of unknown function that does not interact with tRNA^Sec (Fig. 6).¹⁶ Interestingly, the orientation of the C-terminal domain of MJ0158 is similar to that of the SelA C-terminal domain (Fig. 5d and h), suggesting that the change in the domain orientation occurred before SelA acquired its function. Although the N-linker of MJ0158 occupies the similar space to that of SelA, MJ0158 has a Ser residue at the corresponding position of SelA Arg86, which may reflect its functional difference from SelA.

Fig. 6. Structure-based phylogenetic tree of the fold-type-I PLP-dependent enzymes.

SelA and the crystal structures of the fold-type-I PLP enzymes, deposited in the Protein Data Bank, were analyzed. The enzyme names are colored based on their catalysis type: the enzymes that catalyze aminotransfer (AT), α-carboxyl group replacement, α hydrogen replacement, reverse aldol reaction, α,β elimination, β replacement (type 1), β replacement (type 2), α,γ elimination, and γ replacement are colored dark blue, blue, sky blue, blue-violet, magenta, red, orange, spring green, and green, respectively. The enzymes with unknown functions are colored gray.

The structure-based phylogenetic analysis revealed that SelA, SepSecS, and SepCysS³⁴ belong to distinct taxa of the fold-type-I PLP-dependent enzymes. SelA is likely to form an independent group related to the cystathionine γ-synthase (CGS) family. SepCysS is close to the cysteine desulfurase and kynureninase families, while SepSecS also forms another independent group close to the sugar aminotransferase (AT) family.

Since the binding of the A76-Ser moiety of Ser-tRNA is impossible without the decameric assembly (the putative A76 binding site is formed at the dimer-dimer interface),⁷ it is plausible that a dimeric SelA ancestor depended on the phosphate group, and simply converted Sep to Sec without tRNA^Sec discrimination, as in the case of SepSecS (Fig. 7). In fact, the extant SelA can produce Sec-tRNA^Sec from Ser-tRNA^Sec as well as from Sep-tRNA^Sec,⁵ confirming that the Sec synthesis by SelA does not rely on the phosphate group of the substrate aminoacyl moiety. Like SepSecS, the SelA ancestor may have used its selenophosphate binding site to bind the Sep phosphate moiety, to facilitate the elimination of the phosphate group from Sep. In this case, either PSTK or an enzyme with a similar function must have existed for the phosphorylation of Ser and the discrimination of tRNA^Sec from tRNA^Ser. Alternatively, the ability of SepSecS to convert Ser to Sec was lost when PSTK evolved for more efficient Sec production.

Fig. 7. Possible steps of SelA (a) and SepSecS (b) evolution.

The dimeric SelA ancestor accommodated the tRNA^Sec acceptor arm within the cleft between the core and C-terminal domains. The ancestor interacted with the substrate Sep-tRNA^Sec by binding its Sep phosphate group at the selenophosphate (Se-P) binding site. Therefore, the Sep-to-Sec reaction and the tRNA discrimination were dependent on the Sep phosphate group. Tetramerization created the A76 binding pocket, and decamerization stabilized the interactions among intimate dimers. Finally, the acquired N-terminal domain discriminated tRNA^Sec from tRNA^Ser, by interacting with the unique D arm of tRNA^Sec. The A76 binding pocket and the tRNA^Sec discrimination enabled Ser-to-Sec conversion, and thus SelA became independent of the Sep phosphate group. On the other hand, the dimeric SepSecS interacted with the extra arm side of the tRNA^Sec acceptor arm, since it lacks the cleft between the core and C-terminal domains. Tetramerization stabilized the tRNA^Sec binding by interacting with its T and extra arms, without recognizing the unique D-arm structure. Therefore, SepSecS is unable to become independent of the Sep phosphate group of Sep-tRNA^Sec.

Evolving decamerization

In a subsequent evolutionary step, the collaboration of the two SelA dimers could have occurred to generate the A76-binding pocket at the dimer-dimer interface, thereby improving the efficiency and specificity of substrate binding. Nonetheless, in such a tetramer only two of the four catalytic sites would have been functional (the “half-of-the-sites” stage). The relative orientations between the dimers could then be stabilized, by acquiring decameric ring closure involving five dimers. Simultaneously, the full usage of all ten catalytic sites was achieved (Fig. 7a). Interestingly, the non-productive catalytic-site conformation of the dimeric mutant dSelA-ΔN resembles those of the other fold-type-I members (Fig. 3b, c, e to h), and therefore appears to be more primeval. The loop region before α7 interacts with PLP in dSelA-ΔN (Fig. 3b and c), while it forms a β sheet (β6•β7) in the SelA decamer (Fig. 3a). Hence, the catalytic site may have evolved by taking advantage of the decameric quaternary structure. The N-terminal domain was then acquired to increase its affinity for tRNA^Sec, and was further utilized for the discrimination of tRNA^Sec from tRNA^Ser.

Conclusion

The natural decameric arrangement of five SelA dimers allows the formation of ten fully occupied and catalytically functional active sites. Since SelA decamerization is driven by dimer-dimer interactions, disruptive mutations at the interaction interfaces solely yield SelA dimers. In striking contrast to decameric SelA, these dimers are catalytically deficient and exhibit a distinctly distorted active site formation that prevents productive tRNA^Sec coordination. In fact, the dimeric SelA conformationally resembles similar related PLP-enzymes, such as the tetrameric SepSecS. The evolution of the SelA decamer may have been driven by enhanced catalytic efficiency obtained from two sources: First, the decamer allows for an active site conformation that is optimal for direct, tRNA-dependent Ser to Sec conversion. Second, the ten subunit architecture of SelA generates an N-terminal domain with the ability to discriminate tRNA^Sec.

Materials and methods

Protein preparation

The genes encoding the full-length and ΔN SelA genes from A. aeolicus were cloned into the vectors pET25b and pET22b (Novagen), respectively. The full-length SelA used for crystallization was a quadruple mutant, with the non-conserved Lys residues in the loop regions of the N-terminal domain replaced by Ala residues, Lys19Ala-Lys21Ala-Lys46Ala-Lys48Ala (SelA-FL^4KA). The dimerizing quadruple mutation, Tyr220Pro-Asp199Arg-Thr191Tyr-Thr192Tyr, was introduced into SelA-FL^4KA and SelA-ΔN by QuikChange site-directed mutagenesis (Stratagene). The Escherichia coli strain Rosetta 2(DE3) (Stratagene) was transformed with the expression plasmids, and the native and selenomethionine (SeMet)-substituted proteins were overexpressed and purified as described.¹⁷ In order to improve the crystallization efficiency, the lysine residues of dSelA-FL^4KA were methylated with formaldehyde and a dimethylamine-borane complex, as described.¹⁸ Prior to methylation, the protein was diluted with 20 mM Hepes-NaOH buffer (pH 7.5), containing 0.8 M NaCl and 10 mM 2-mercaptoethanol (2-ME).

Crystallization and X-ray diffraction data collection

The SelA proteins were crystallized at 20°C by the sitting-drop vapor diffusion method, by mixing 0.75 µl of the sample with 0.75 µl of reservoir solutions. Prior to crystallization, dSelA-FL^4KA (SeMet substitution, Lys methylation) and dSelA-ΔN (SeMet substitution, without methylation) were dialyzed against 20 mM Tris-HCl buffer (pH 7.5), containing 200 mM NaCl and 10 mM 2-ME, and were concentrated to 9.6 and 7.8 mg/ml, respectively.

Crystal Screen 2, Natrix (Hampton Research), Wizard I, and Wizard II (Emerald Bio-Science) screening kits were used for the initial screening of the crystallization conditions. The optimized reservoir solutions were 100 mM trisodium citrate-HCl buffer (pH 5.6), containing 580–590 mM trisodium citrate, 100 mM L-serine, and 100 mM Na₂S₂O₃ for dSelA-FL^4KA, and 100 mM sodium MES-HCl buffer (pH 6.5), containing 2.5% polyethylene glycol (PEG) 4,000, 150 mM Na₂S₂O₃, and 70 mM NaNO₃ for dSelA-ΔN.

The crystals thus obtained were transferred into cryo-protective solutions, composed of 100 mM trisodium citrate-HCl buffer (pH 5.6), containing 1.3 M trisodium citrate, 75 mM L-serine, and 75 mM Na₂S₂O₃ for dSelA-FL^4KA, and 100 mM sodium MES-HCl buffer (pH 6.5), containing 30% dimethyl sulfoxide, 10% PEG 4,000, 50 mM Na₂S₂O₃, and 70 mM NaNO₃ for dSelA-ΔN, and were flash-cooled in a cryo-N₂ stream. X-ray diffraction data were collected at 90 K, by using synchrotron radiation at the BL41XU of SPring-8 (Harima, Japan). The data were processed with the HKL2000 program.¹⁹ The statistics of the diffraction-data collection are listed in Table 2.

Table 2.

Data collection and refinement statistics.

	dSelA-FL4KA (SeMet)	dSelA-ΔN (SeMet)
Lysmethylation	+	−
Data collection
Beam line	SPring-8 BL41XU	SPring-8 BL41XU
Wavelength (Å)	0.97915	0.97915
Space group	I422	P21
Cellparameters
a (Å)	144.1	59.2
b (Å)	144.1	81.3
c (Å)	273.4	95.5
β (°)		92.2
Resolution (Å)	50.0–3.35 (3.47–3.35)a	50.0–2.40 (2.49–2.40)a
Unique reflections	21,096 (2066)a	35,457 (3520)a
Completeness (%)	99.7 (100.0)a	99.6 (100.0)a
Redundancy	6.0 (6.1) a	3.8 (3.8)a
Rsymb	0.102 (0.712)a	0.082 (0.561)a
I/σ(I)	17.2 (2.75)a	15.9 (2.40)a
Structure refinement
Working-set reflections	19,970	33,593
Test-set reflections	1062	1770
Resolution (Å)	50.0–3.35	50.0–2.40
Number of SelA subunits	2 (one dimer)	2 (one dimer)
Number of protein atoms	7035	6018
Number of PLP	0	2
Number of ions	4 (thiosulfate)	5 (thiosulfate)
Number of solvent molecules	0	153
Rwork/Rfreec	0.194/0.250	0.180/0.230
Average B-factor (Å2)
Overall	144.3	64.4
Protein	144.2	64.6
PLPs	—	49.8
Ion	162.4	79.0
Solvent	—	56.7
RMSD bond lengths (Å)	0.009	0.008
RMSD bond angles (°)	1.27	1.12

^aThe statistics in the highest-resolution shell are given in parentheses.

^bR_sym = ∑_hkl∑_i[|I_i(hkl) − 〈I(hkl)〉|]/∑_hkl∑_i[I_i(hkl)], where I_i(hkl) is the intensity of the ith measurement of hkl and 〈I(hkl)〉 is the average value of I_i(hkl) for all ith measurements.

^cR_{work, free} = ∑_hkl(‖F_o| − k|F_c‖)/∑_hkl(|F_o|), where R_work and R_free are calculated using the working-set and test-set reflections (5% of the total reflections), respectively.

Structure determination and refinement

The structures of the dimeric SelAs, dSelA-FL^4KA and dSelA-ΔN, were solved by the molecular replacement method, using the Phaser program.²⁰ The search model was the SelA-ΔN structure. The Coot program²¹ was used for manual fitting of the models to the electron density map. The structures were refined against the diffraction data by using the CNS²² and Phenix²³ programs, with iterative cycles of positional and temperature-factor refinements (Table 2).

Production and purification of E. coli SelA variants

Mutations were introduced by site-directed mutagenesis (Quikchange^®, Agilent) to E. coli K12 selA, from the ASKA E. coli K12 ORF library (GFP-minus), cloned in the pC24N vector²⁴. E. coli BL21 cells were transformed with the expression plasmids. For protein overproduction, the cells were grown at 25°C in LB selective medium, supplemented with 50 µM isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells sedimented from over-night cultures were resuspended in lysis buffer (50 mM KH₂PO₄, pH 8, 300 mM NaCl, 15% glycerol, 10 mM imidazole) and disrupted by sonication. After centrifugation (50,000 g, 45 min, 4°C), the clarified lysates were subjected to immobilized-metal affinity chromatography (IMAC), using Ni-NTA affinity resin (Qiagen) on gravity-flow columns. The immobilized 6xHis-SelA fusion proteins were washed with lysis buffer supplemented with 40 mM imidazole, and SelA was eluted with 240 mM imidazole in lysis buffer. The excess imidazole was removed by dialysis (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 15% glycerol), and the purified SelA variants were analyzed by SDS-PAGE and stored at −80°C. The amounts of protein-bound PLP in the SelA variants were determined by UV/vis spectroscopy at 420 nm and comparison to a PLP standard curve (0–2 mM).

E. coli SelA in vivo activity assays

For in vivo SelA assays, a marker-less E. coli selA deletion strain was used. The initial selA deletion strain JS1 was constructed by the replacement of the selA gene with a kanamycin cassette in E. coli strain BW25113, as described²⁵, and the genomic insertion of T7 polymerase by P1 transduction⁴. The kanamycin resistance marker of E. coli strain JS1 was then excised by FLP recombinase-mediated homologous recombination between the FRT sites flanking the kanamycin resistance cassette, upon transformation of the JS1 strain with the plasmid pCP20²⁶, resulting in the marker-less E. coli ΔselA strain JS2. E. coli ΔselA was then transformed with the plasmids encoding the E. coli SelA variants and grown on LB selective medium. Overnight cultures of these clones were plated on selective LB agar plates supplemented with 10 µM IPTG, 1 µM Na₂MoO₄, 1 µM Na₂SeO₃ and 50 mM sodium formate, as described previously⁴, and grown anaerobically at 37°C over-night. For the oxygen-sensitive benzyl viologen assay, the plates were transferred to an anaerobic chamber (90% N₂, 5% H₂, 5% CO₂) and overlaid with top agar containing 1 mg/mL benzyl viologen, 250 mM sodium formate and 25 mM KH₂PO₄/K₂HPO₄, pH 7.0. Wild type SelA served as a positive control. The benzyl viologen assay allows the colorimetric monitoring of SelA activity, by the formation of purple-colored reduced benzyl viologen. Benzyl viologen reduction is catalyzed by FDH_H, which requires Sec insertion for catalytic activity. The Sec formation, finally, is dependent on the SelA activity.

Structure-based phylogenetic tree

The STAMP program²⁷ in the Multiseq module of VMD²⁸ was used to superpose and calculate the structural similarity, Q_H, among the coordinates of SelA-ΔN and the other fold-type-I PLP-dependent enzymes obtained from the Protein Data Bank. The enzymes with >50% amino-acid sequence identity were removed, and thus the core domains of 187 coordinates, including the coordinates of both chains A and B of the hetero-dimeric glycine decarboxylase (PDB ID: 1WYT), were superposed. The other 185 enzymes were homo-oligomeric ones. The structural similarity Q_H was calculated²⁹, and the neighbor-joining tree, based on the Q_H values, was drawn using the SplitsTree program³⁰.

Abbreviations

AHBA

5-amino-3-hydroxybenzoate

AT

aminotransferase

CBL

cystathionine β-lyase

CBS

cystathionine β-synthase

CGS

cystathionine γ-synthase

CysS

cysteine synthase

2-ME

2-mercaptoethanol

PEG

polyethylene glycol

PLP

pyridoxal 5′-phosphate

PSTK

O-phosphoseryl-tRNA^Sec kinase

Sec

selenocysteine

SeMet

selenomethionine

SerRS

seryl-tRNA synthetase