From the Shanghai Center for Plant Stress Biology and Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
J Biol Chem. 2013 Nov 1;288(44):31540-8. doi: 10.1074/jbc.M113.496828. Epub 2013 Sep 18.
Pentatricopeptide repeat (PPR) proteins are sequence-specific RNA-binding proteins that form a pervasive family of proteins conserved in yeast, plants, and humans. The plant PPR proteins are grouped mainly into the P and PLS classes. Here, we report the crystal structure of a PLS-class PPR protein from Arabidopsis thaliana called THA8L (THA8-like) at 2.0 Å. THA8L resembles THA8 (thylakoid assembly 8), a protein that is required for the splicing of specific group II introns of genes involved in biogenesis of chloroplast thylakoid membranes. The THA8L structure contains three P-type PPR motifs flanked by one L-type motif and one S-type motif. We identified several putative THA8L-binding sites, enriched with purine sequences, in the group II introns. Importantly, THA8L has strong binding preference for single-stranded RNA over single-stranded DNA or double-stranded RNA. Structural analysis revealed that THA8L contains two extensive patches of positively charged residues next to the residues that are proposed to comprise the RNA-binding codes. Mutations in these two positively charged patches greatly reduced THA8L RNA-binding activity. On the basis of these data, we constructed a model of THA8L-RNA binding that is dependent on two forces: one is the interaction between nucleotide bases and specific amino acids in the PPR motifs (codes), and the other is the interaction between the negatively charged RNA backbone and positively charged residues of PPR motifs. Together, these results further our understanding of the mechanism of PPR protein-RNA interactions.
五肽重复（PPR）蛋白是序列特异性的 RNA 结合蛋白，形成了一个在酵母、植物和人类中保守的广泛存在的蛋白质家族。植物 PPR 蛋白主要分为 P 和 PLS 两类。在这里，我们报告了来自拟南芥的 PLS 类 PPR 蛋白 THA8L（THA8 样）的晶体结构，分辨率为 2.0Å。THA8L 类似于 THA8（类囊体组装 8），这是一种蛋白质，对于参与叶绿体类囊体膜生物发生的基因的特定组 II 内含子的剪接是必需的。THA8L 结构包含三个 P 型 PPR 基序，两侧分别为一个 L 型基序和一个 S 型基序。我们在组 II 内含子中鉴定了几个富含嘌呤序列的推定 THA8L 结合位点。重要的是，THA8L 对单链 RNA 的结合偏好强于单链 DNA 或双链 RNA。结构分析表明，THA8L 在拟议的 RNA 结合码的残基旁边包含两个广泛的带正电荷的残基。这两个带正电荷的斑块中的突变大大降低了 THA8L RNA 结合活性。基于这些数据，我们构建了一个 THA8L-RNA 结合模型，该模型依赖于两种力：一种是核苷酸碱基与 PPR 基序（码）中特定氨基酸之间的相互作用，另一种是负电荷 RNA 骨架与 PPR 基序中带正电荷残基之间的相互作用。总之，这些结果进一步了解了 PPR 蛋白-RNA 相互作用的机制。
