1918–1926 Nucleic Acids Research, 2015, Vol. 43, No. 3 Published online 21 January 2015 doi: 10.1093/nar/gkv027 The solution structure of the pentatricopeptide repeat protein PPR10 upon binding atpH RNA Benjamin S. Gully1, Nathan Cowieson2, Will A. Stanley1,3, Kate Shearston1,3, Ian D. Small3, Alice Barkan4 and Charles S. Bond1,*
1School of Chemistry and Biochemistry, The University of Western Australia, Crawley, Western Australia, Australia, 2SAXSWAXS beamline, Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria, Australia, 3Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Western Australia, Australia and 4Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA
Received May 29, 2014; Revised December 21, 2014; Accepted January 09, 2015
ABSTRACT (3) and translational control (10–14). Mutations can result in cytoplasmic male sterility (15,16), impaired seed devel- The pentatricopeptide repeat (PPR) protein fam- opment (17) and diverse embryonic defects (18). ily is a large family of RNA-binding proteins that PPR proteins can be broadly divided into two families, is characterized by tandem arrays of a degener- those consisting of tandem arrays of canonical P-class mo- ate 35-amino-acid motif which form an ␣-solenoid tifs and those composed of repeats of triplets of P-, L- and structure. PPR proteins influence the editing, splic- S-class motifs (3). Regardless of repeat class, conservation ing, translation and stability of specific RNAs in of key amino acids from each motif has been correlated with mitochondria and chloroplasts. Zea mays PPR10 the sequence of known RNA targets to establish a statisti- is amongst the best studied PPR proteins, where cally and experimentally validated predictive code for RNA sequence-specific binding to two RNA transcripts, recognition by PPR proteins (19–21). This code is consis- atpH and psaJ, has been demonstrated to follow a tent with a modular interaction where a base is coordinated by the sixth residue of one PPR repeat, and the first residue recognition code where the identity of two amino of the next repeat. Such a modular, predictable, mode of acids per repeat determines the base-specificity. single-stranded RNA binding makes PPR proteins a po- A recently solved ZmPPR10:psaJ complex crystal tential scaffold for the design of biotechnologically useful structure suggested a homodimeric complex with proteins (22,23). considerably fewer sequence-specific protein–RNA Predictions of an ␣-solenoid tertiary structure for PPR contacts than inferred previously. Here we describe tracts (1,24) have been confirmed recently by a number of the solution structure of the ZmPPR10:atpH com- crystal structures including a segment of the mitochondrial plex using size-exclusion chromatography-coupled RNA polymerase (25), Arabidopsis proteinaceous ribonu- synchrotron small-angle X-ray scattering (SEC-SY- clease P (26), the P-class PPR protein THA8L (27) and a SAXS). Our results support prior evidence that synthetic consensus P-class PPR protein (28). Crystal struc- PPR10 binds RNA as a monomer, and that it does tures of two PPR proteins, Brachypodium THA8 (29)and Zea mays PPR10 (ZmPPR10) (30), in complex with RNA so in a manner that is commensurate with a canoni- have partially confirmed the modular RNA-binding mode, cal and predictable RNA-binding mode across much but revealed non-modular and idiosyncratic interactions of the RNA–protein interface. that had not been anticipated based on prior data. In addi- tion, these structures implicate a dimeric quaternary struc- INTRODUCTION ture in RNA binding. ZmPPR10 localizes to plastids where it binds cis- The pentatricopeptide repeat (PPR) superfamily (1)ischar- elements of two polycistronic transcripts (6,13), the mini- acterized by a degenerate 35 amino acid repeat and is spe- mal binding sites identified as 17 and 18 nucleotide tracts in cific to eukaryotes although most numerous and varied in the atpI-atpH and psaJ-rpl33 intercistronic regions. PPR10 the plant kingdom (2), with over 400 identified in Arabidop- binding was shown to stabilize the RNA in vivo, protect- sis thaliana (3). PPR proteins function in RNA stabiliza- ing the transcripts from exoribonucleases in either direc- tion (4–6), editing (7), maturation and post-transcriptional tion, and to activate mRNA translation by remodeling lo- modifications (8,9) and are essential for organelle biogenesis
*To whom correspondence should be addressed. Tel: +61 8 6488 4406; Fax: +61 8 6488 7330; Email: [email protected] Present address: Will A. Stanley, Northern Institute for Cancer Research, University of Newcastle upon Tyne, UK.