Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL60607, USA.
Nucleic Acids Res. 2023 May 8;51(8):4012-4026. doi: 10.1093/nar/gkad182.
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. 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-, TΨC-, and variable arms of tRNASec and helices α1 and α14 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.
O-磷酸丝氨酰-tRNA（Sec）硒转移酶（SepSecS）催化古菌和真核生物中硒代半胱氨酸（Sec）合成的终末步骤。Sec 合成机制如何识别和区分 tRNASec 和 tRNA 池对于硒蛋白组的完整性至关重要。先前，我们提出 SepSecS 采用了一种具有预催化能力的构象。在此，我们使用高分辨率 X 射线晶体学可视化了人类 SepSecS 中依赖于 tRNA 的构象变化，这可能是实现催化能力的先决条件。我们表明，tRNASec 的结合组织了催化单体的活性位点，同时稳定了非催化单体的 N-和 C-末端。大阴离子与催化槽的结合可能进一步优化催化位点以进行底物结合和催化。我们的生化和突变分析表明，产生活性 SepSecS•tRNASec 复合物的形成主要是由 tRNASec 的接受臂、TΨC-臂和可变臂与 SepSecS 的α1 和α14 螺旋之间的静电相互作用驱动的，是焓驱动的。tRNA 依赖性激活 SepSecS 的详细可视化提供了一个结构基础，用于修正古菌和真核生物中 Sec 形成的末端反应模型。
