Decameric SelA•tRNA ^Sec Ring Structure Reveals Mechanism of Bacterial Selenocysteine Formation

Abstract

The 21st amino acid, selenocysteine (Sec), is synthesized on its cognate transfer RNA (tRNASec). In bacteria, SelA synthesizes Sec from Ser-tRNASec, whereas in archaea and eukaryotes SepSecS forms Sec from phosphoserine (Sep) acylated to tRNASec. We determined the crystal structures of Aquifex aeolicus SelA complexes, which revealed a ring-shaped homodecamer that binds 10 tRNASec molecules, each interacting with four SelA subunits. The SelA N-terminal domain binds the tRNASec-specific D-arm structure, thereby discriminating Ser-tRNASec from Ser-tRNASer. A large cleft is created between two subunits and accommodates the 3′-terminal region of Ser-tRNASec. The SelA structures together with in vivo and in vitro enzyme assays show decamerization to be essential for SelA function. SelA catalyzes pyridoxal 5′-phosphate–dependent Sec formation involving Arg residues nonhomologous to those in SepSecS. Different protein architecture and substrate coordination of the bacterial enzyme provide structural evidence for independent evolution of the two Sec synthesis systems present in nature.

Selenoproteins: Molecular Pathways and Physiological Roles

“…This has been further confirmed by recent crystallographic studies that revealed an interaction of the variable stem of tRNA [Ser]Sec with the NH 2 -terminal domain of SerS (156,157). Moreover, the orientation of the extended variable arm in the tertiary structure of tRNA [Ser]Sec is nearly identical to that of tRNA Ser , suggesting that it is recognized by SerS in the same manner, and is distinguished from the other tRNAs (155). However, the overall contribution of SerS to charging tRNA [Ser]Sec accounts for only …”

Formation of a Ternary Complex for Selenocysteine Biosynthesis in Bacteria

“…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) (Fig. 2, A and B).…”

“…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 7-12-nucleotide 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-seryltRNA Sec , aminoacylated with serine by seryl-tRNA synthetase (SerRS), to L-selenocysteyl-tRNA Sec in a reaction catalyzed by selenocysteine synthase (E.C.…”

“…2 To whom correspondence should be addressed: Physics and Informatics Dept., Physics Institute of Sao Carlos, University of Sao Paulo -USP, Joao Dagnone Av, 1100, Jardim Santa Angelina, CEP 13563-120, Sao Carlos, SP, Brazil. 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.…”

Eine einfache Methode zur Produktion von Selenoproteinen

“…[9] Dabei gingen wir davon aus, dass Modifikationen, welche die Protein-Protein-Interaktion der beiden Domänen beeinflussen, die Orientierung der N-terminalen Domäne verändern würden und folglich die Umwandlung von Ser zu Sec auf der allo-tRNA verbessern könnten. Auf Grundlage der Proteinstruktur eines Komplexes aus SelA und tRNA Sec[9] modifizierten wir die Aminosäurereste Pro68, Leu69, Gln72 und Cys173 von As -SelA, die Ile25, Tyr26, Lys29 und Glu129 von SelA aus Aquifex aeolicus entsprechen (Abbildungen 4 A und S8). Zum Screening der As -SelA-Mutantenbibliothek verwendeten wir eine NMC-A-β-Lac-tamasevariante, [6c] deren essenzielle Disulfidbrücke in E.-coli -C321.ΔA.opt Δ selAB durch eine Diselenitbrücke ersetzt werden muss (Abbildung 4 B).…”