Structural And Biochemical Study Of PPR Proteins

Pentatricopeptide repeat(PPR) proteins, particularly abundant in chloroplasts and mitochondria of terrestrial plants, represent a large number of sequence-specific RNA binding proteins that are involved in diverse functional aspects of organelle RNA metabolisms. PPR10 from maize chloroplast can bind two native single-stranded RNA elements atpH and psaJ. Despite extensive biochemical characterizations and rigorous functional identifications for PPR10, the structural basis for sequence-specific RNA recognition by PPR proteins remains unknown. PPR proteins contain multiple repeats. Through residues 2, 5, and 35, an intact repeat retains the capability of single-stranded RNA recognition in a modular fashion. Previous computational and biochemical analyses proposed RNA recognition code by PPR proteins. Structural elucidation and biochemical corroboration of RNA recognition code still waits further appraisals.Initially, we have sought to determine the crystal structure of PPR10. We obtained PPR10 protein by recombinant expression. Numerous methods of crystallization trials were performed. Finally, we determined the crystal structure of PPR10 at 2.85 ?.Moreover, we successfully reconstituted PPR10 and its RNA target in vitro. After considerable protein engineering effort, we eventually obtained the crystal structure of PPR10 in complex with psaJ RNA element at 2.45 ?. In the crystal, antiparallel dimer formation is observed and RNA recognition code is validated. For RNA specificity determinant residues at 2, 5, and 35 within a repeat, their respective roles are illustrated.Furthermore, we proceeded to reveal determining factor for PPR10 dimerization. We discovered that the dimerization state formation and binding affinity for PPR10 with RNA is regulated by amino terminal length. Simultaneously, we found the key residue mutation can also determine the dimerization state of PPR10 with RNA and affect PPR10 binding property with RNA.Additionally, we designed the novel PPR protein scaffold consisting of ten consecutive repeats. Consistent with the RNA recognition code, the designer PPR protein can specifically bind the target RNA element with high affinity.