Architecture of the Terminal Complex of Selenocysteine Synthesis in Humans.

O-phosphoseryl-tRNASec selenium transferase (SepSecS) catalyzes the terminal step of selenocysteine (Sec) synthesis in archaea and eukaryotes. How the Sec synthetic machinery recognizes and discriminates tRNASec from the tRNA pool is essential to the integrity of the selenoproteome. Previously, we suggested that SepSecS adopts a competent conformation that is pre-ordered for catalysis. Herein, using high-resolution X-ray crystallography, we visualized tRNA-dependent conformational changes in human SepSecS that may be a prerequisite for achieving catalytic competency. We show that tRNASec binding organizes the active sites of the catalytic protomer, while stabilizing the N- and C-termini of the non-catalytic protomer. Our results suggest that a C-terminal helix regulates the overall architecture of the SepSecS•tRNASec complex by docking in the tRNA binding sites of the non-catalytic protomer, precluding tRNA binding. Binding of large anions to the catalytic groove may further optimize the catalytic site for substrate binding and catalysis. Our biochemical and mutational analyses demonstrate that productive SepSecS•tRNASec complex formation is enthalpically driven and primarily governed by electrostatic interactions between the acceptor-, TpsiC-, and variable arms of tRNASec and helices alpha1 and alpha14 of SepSecS. The detailed visualization of the tRNA-dependent activation of SepSecS provides a structural basis for a revised model of the terminal reaction of Sec formation in archaea and eukaryotes.