Vasu Kommireddy, Ramachandiran Iyappan, Terenzi Fulvia, Khan Debjit, China Arnab, Khan Krishnendu, Chechi Aayushi, Baleanu-Gogonea Camelia, Gogonea Valentin, Fox Paul L
Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA.
Department of Chemistry, Cleveland State University, Cleveland, OH, USA.
iScience. 2021 Feb 20;24(3):102215. doi: 10.1016/j.isci.2021.102215. eCollection 2021 Mar 19.
Aminoacyl-tRNA synthetases (AARS) participate in decoding the genome by catalyzing conjugation of amino acids to their cognate tRNAs. During evolution, biochemical and environmental conditions markedly influenced the sequence and structure of the 20 AARSs, revealing adaptations dictating canonical and orthogonal activities. Here, we investigate the function of the appended Zn-binding domain (ZBD) in the bifunctional AARS, glutamyl-prolyl-tRNA synthetase (GluProRS). We developed GluProRS mutant mice by CRISPR-Cas9 with a deletion of 29 C-terminal amino acids, including two of four Zn-coordinating cysteines. Homozygous ZBD mutant mice die before embryonic day 12.5, but heterozygous mice are healthy. ZBD disruption profoundly reduces GluProRS canonical function by dual mechanisms: it induces rapid proteasomal degradation of the protein and inhibits ProRS aminoacylation activity, likely by sub-optimal positioning of ATP in the spatially adjacent catalytic domain. Collectively, our studies reveal the ZBD as a critical determinant of ProRS activity and GluProRS stability and .
氨酰-tRNA合成酶（AARS）通过催化氨基酸与其同源tRNA的结合参与基因组解码。在进化过程中，生化和环境条件显著影响了20种AARS的序列和结构，揭示了决定经典和正交活性的适应性变化。在此，我们研究了双功能AARS——谷氨酰-脯氨酰-tRNA合成酶（GluProRS）中附加的锌结合结构域（ZBD）的功能。我们通过CRISPR-Cas9技术构建了GluProRS突变小鼠，缺失了29个C末端氨基酸，包括四个锌配位半胱氨酸中的两个。纯合ZBD突变小鼠在胚胎第12.5天之前死亡，但杂合小鼠健康。ZBD破坏通过双重机制显著降低GluProRS的经典功能：它诱导蛋白质的快速蛋白酶体降解，并抑制ProRS氨酰化活性，这可能是由于ATP在空间相邻催化结构域中的定位不理想所致。总的来说，我们的研究揭示了ZBD是ProRS活性和GluProRS稳定性的关键决定因素。
