Structural Asymmetry of the Terminal Catalytic Complex in Selenocysteine Synthesis [Protein Structure and Folding]

September 4th, 2014 by French, R. L., Gupta, N., Copeland, P. R., Simonović, M.

Selenocysteine (Sec), the 21st amino acid, is synthesized from a serine precursor in a series of reactions that require selenocysteine tRNA (tRNASec). In archaea and eukaryotes, O-phosphoseryl-tRNASec:selenocysteinyl-tRNASec synthase (SepSecS) catalyzes the terminal synthetic reaction during which the phosphoseryl intermediate is converted into the selenocysteinyl moiety while attached to tRNASec. We have previously shown that only the SepSecS tetramer is capable of binding to and recognizing the distinct fold of tRNASec. Because only two of the four tRNA-binding sites were occupied in the crystal form, a question was raised if the observed arrangement and architecture faithfully recapitulated the physiologically relevant ribonucleoprotein complex important for selenoprotein formation. Herein, we determined the stoichiometry of the human terminal synthetic complex of selenocysteine by using small-angle X-ray scattering, multi-angle light scattering, and analytical ultracentrifugation. In addition, we provided the first estimate of the ratio between SepSecS and tRNASec in vivo. We show that SepSecS preferentially binds one or two tRNASec molecules at a time, and that the enzyme is present in large molar excess over the substrate tRNA in vivo. Moreover, we show that in a complex between SepSecS and two tRNAs, one enzyme homodimer plays a role of the non-catalytic unit that positions CCA ends of two tRNASec molecules into the active-site grooves of the other, catalytic, homodimer. Finally, our results demonstrate the previously determined crystal structure represents the physiologically and catalytically relevant complex, and suggest that allosteric regulation of SepSecS might play an important role in regulation of selenocysteine and selenoprotein synthesis.