School of Life Sciences, Key Laboratory of Cell Activities and Stress Adaptation of the Ministry of Education, Lanzhou University, Lanzhou 730000, China.
Nucleic Acids Res. 2024 May 8;52(8):4523-4540. doi: 10.1093/nar/gkae179.
In archaea and eukaryotes, the evolutionarily conserved KEOPS is composed of four core subunits-Kae1, Bud32, Cgi121 and Pcc1, and a fifth Gon7/Pcc2 that is found in fungi and metazoa. KEOPS cooperates with Sua5/YRDC to catalyze the biosynthesis of tRNA N6-threonylcarbamoyladenosine (t6A), an essential modification needed for fitness of cellular organisms. Biochemical and structural characterizations of KEOPSs from archaea, yeast and humans have determined a t6A-catalytic role for Kae1 and auxiliary roles for other subunits. However, the precise molecular workings of KEOPSs still remain poorly understood. Here, we investigated the biochemical functions of A. thaliana KEOPS and determined a cryo-EM structure of A. thaliana KEOPS dimer. We show that A. thaliana KEOPS is composed of KAE1, BUD32, CGI121 and PCC1, which adopts a conserved overall arrangement. PCC1 dimerization leads to a KEOPS dimer that is needed for an active t6A-catalytic KEOPS-tRNA assembly. BUD32 participates in direct binding of tRNA to KEOPS and modulates the t6A-catalytic activity of KEOPS via its C-terminal tail and ATP to ADP hydrolysis. CGI121 promotes the binding of tRNA to KEOPS and potentiates the t6A-catalytic activity of KEOPS. These data and findings provide insights into mechanistic understanding of KEOPS machineries.
在古菌和真核生物中，进化上保守的 KEOPS 由四个核心亚基-Kae1、Bud32、Cgi121 和 Pcc1 以及第五个 Gon7/Pcc2 组成，后者仅存在于真菌和后生动物中。KEOPS 与 Sua5/YRDC 合作催化 tRNA N6-硫代胞苷酰腺苷（t6A）的生物合成，这是细胞生物适应性所必需的关键修饰。对古菌、酵母和人类的 KEOPS 的生化和结构特征进行了研究，确定了 Kae1 的 t6A 催化作用以及其他亚基的辅助作用。然而，KEOPS 的精确分子机制仍知之甚少。在这里，我们研究了拟南芥 KEOPS 的生化功能，并确定了拟南芥 KEOPS 二聚体的 cryo-EM 结构。我们表明，拟南芥 KEOPS 由 KAE1、BUD32、CGI121 和 PCC1 组成，采用保守的整体排列。PCC1 二聚化导致 KEOPS 二聚体的形成，这对于活性 t6A 催化 KEOPS-tRNA 组装是必需的。BUD32 参与 tRNA 与 KEOPS 的直接结合，并通过其 C 末端尾巴和 ATP 到 ADP 水解来调节 KEOPS 的 t6A 催化活性。CGI121 促进 tRNA 与 KEOPS 的结合，并增强 KEOPS 的 t6A 催化活性。这些数据和发现为 KEOPS 机制的机制理解提供了新的见解。
