The human SepSecS-tRNASec complex reveals the mechanism of selenocysteine formation

Abstract

Selenocysteine is the only genetically encoded amino acid in humans whose biosynthesis occurs on its cognate transfer RNA (tRNA). O-Phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS) catalyzes the final step of selenocysteine formation by a poorly understood tRNA-dependent mechanism. The crystal structure of human tRNA(Sec) in complex with SepSecS, phosphoserine, and thiophosphate, together with in vivo and in vitro enzyme assays, supports a pyridoxal phosphate-dependent mechanism of Sec-tRNA(Sec) formation. Two tRNA(Sec) molecules, with a fold distinct from other canonical tRNAs, bind to each SepSecS tetramer through their 13-base pair acceptor-TPsiC arm (where Psi indicates pseudouridine). The tRNA binding is likely to induce a conformational change in the enzyme's active site that allows a phosphoserine covalently attached to tRNA(Sec), but not free phosphoserine, to be oriented properly for the reaction to occur.

Fig. 1

Structure of human SepSecS in complex with unacylated tRNA^Sec. (A) Surface representation of the physiological complex of SepSecS with tRNA^Sec. The subunits of the catalytic dimer are dark and light blue, those of the noncatalytic dimer are dark and light red; the backbone and the bases of tRNA^Sec are green and gray, respectively. (B) The catalytic dimer interacts with the acceptor arm of tRNA^Sec through helices α14 and α15 (blue). The α1 helix (red) of the noncatalytic dimer interacts with the rest of the acceptor-TΨC arm. The regions of tRNA^Sec that interact with SepSecS are shown in orange; the rest is green. One tRNA^Sec molecule is shown for clarity. (C) Interactions between the discriminator base G73 and the conserved Arg³⁹⁸ in the α14-β11 loop. The protein side chains are gold, and tRNA^Sec is green.

Fig. 2

Structure of human tRNA^Sec. (A) Ribbon diagram of the human tRNA^Sec molecule observed in complex with SepSecS. The major structural elements are colored as follows: the acceptor arm is red, the D-arm is blue, the anticodon arm is light blue, the variable arm is orange, and the TΨC arm is dark red. (B) The view is rotated by ~90° clockwise around the vertical axis. (C) Secondary structure diagram of human tRNA^Sec derived from the crystal structure.

Fig. 3

Ligand binding to the active sites in the SepSecS-tRNA^Sec complex. In (A), (C), (D), (E), and (F), the catalytic PLP-monomer is purple, the P-loop monomer is light blue, Sep is gold, tRNA is green, and PLP is magenta. In (A) and (B), the unbiased omit electron density map (green mesh) is contoured at 3.5 σ. (A) Phosphoserine binds to the catalytic sites in two orientations (Sep^P and Sep^N). (B) Thiop (orange) binds only to the non-catalytic site. The PLP-monomer is brown, and the P-loop monomer is pink. (C) The Sep^N amino group is ~12 Å away from the Schiff base. Arg⁹⁷, Gln¹⁰⁵, and Arg³¹³ coordinate phosphate, and the carboxyl group interacts with Lys¹⁷³. (D) The amino group of Sep^P interacts with Gln¹⁷² and is 3.5 Å away from the Schiff base. The carboxyl group interacts with Gln¹⁷², whereas Ser⁹⁸, Gln¹⁰⁵, and Arg³¹³ coordinate phosphate. (E) The P-loop adopts different conformation after tRNA^Sec binding. The noncatalytic dimer is pink, the catalytic dimer is light blue. Steric clashes between the noncatalytic P-loop and Sep^N are shown (double arrow). (F) A model of CCA-Sep^P (gold) in the catalytic active site. A76 binds between the side chains of Arg⁹⁷ and Lys¹⁷³, whereas C75 interacts with Arg⁹⁷.

Fig. 4

The PLP-dependent mechanism of Sep to Sec conversion. (A) The phosphoseryl moiety of Sep-tRNA^Sec is bound to the active site similar to Sep^P. The amino group is oriented for attack on the Schiff base, whereas the phosphoryl group is stabilized by the side chains of Ser⁹⁸, Gln¹⁰⁵, and Arg³¹³. Hydrogen bonds are shown in dashed lines. (B) After the formation of the external aldimine, the side chain of Lys²⁸⁴ rearranges and abstracts the Cα proton from Sep. The protonated pyridine ring of PLP stabilizes the carbanion. (C) Electron delocalization leads to a rapid β-elimination of phosphate and to the formation of dehydroalanyl-tRNA^Sec. Free phosphate dissociates, and selenophosphate binds to the active site. (D) An unidentified base (^–B) activates water that hydrolyzes selenophosphate. Free phosphate dissociates again, and selenium attacks the dehydroalanyl-tRNA^Sec. Lys²⁸⁴ returns the proton to the Cα carbon, and the selenocysteinyl moiety is formed. (E) The reaction of reverse transaldimination is shown. Lys²⁸⁴ forms the Schiff base, with PLP leading to a release of the oxidized form of Sec-tRNA^Sec (red box). (F) The free amino group of Sec-tRNA^Sec is protonated, and the active site of SepSecS is regenerated.