Oral Presentation The 35th Biennial Conference of the Society of Crystallographers in Australia and New Zealand 2024 (Crystal 35)

Decoding the Mysteries of S-class PPR Proteins: A Molecular Detective Story (#105)

Anuradha Pullakhandam 1 2 , Crystal Cooper 3 , Michael Landsberg 4 , Charlie Bond 1 , Ian Small 1 2
  1. School of Molecular Sciences, The University of Western Australia, Crawley, WA, Australia
  2. ARC centre of excellence for plant energy biology, The University of Western Australia, Crawley, WA, Australia
  3. Centre for Microscopy Characterisation and Analysis, The Univeristy of Western Australia, Crawley, WA, Australia
  4. School of Chemistry and Molecular Sciences, The University of Queensland, Brisbane St Lucia, Queensland, Australia

RNA editing is a process of altering the nucleotide bases within an RNA transcript, resulting in a chemically modified RNA molecule distinct from its genomic DNA (Benne, 1989). It is mainly carried out through base substitution, insertion, or deletion (Benne, 1989). In plants, Pentatricopeptide (PPR) proteins are well-known and involved in the RNA transcript's single-base editing (Okuda et al., 2009). Thus far, more than a thousand editing sites have been unravelled in Club Moss and Hornworts (Lenz and Knoop, 2013), and it has been established that the conserved C-terminal DYW domain causes the site-specific RNA editing activity(Cheng et al., 2016; Lenz and Knoop, 2013). This study focuses on revealing the structural architecture, flexibility, dynamics and molecular mechanism of the intact S-class PPR-DYW editing factors. We also aim to understand the RNA binding specificity in S-class PPR proteins and how it impacts the conformation of the protein. To do this, designer PPR proteins were expressed and purified using Ni2+ affinity Chromatography and Size Exclusion Chromatography. Computational methods (Alpha Fold) and biophysical techniques such as Small-angle X-ray Scattering (SAXS) and Cryo-Electron Microscopy were used to elucidate the structure of S-class PPR editing factors. SDS-PAGE gel electrophoresis of SEC fractions showed a single band approximately at 100 kDa corresponding to the theoretical molecular weight of the protein (105 kDa). Furthermore, this was also confirmed through the SAXS data which ~ 104.9 kDa (confidence interval of 95.8 to 111.2). The estimated molecular weight of the complex is 108 kDa, and the SAXS data showed a confidence of 101 kDa (confidence interval ranging from 95.8 to 121.5 kDa), within the expected range. The Dmax of the protein alone is 180 Å, and its complex is 150 Å. The pairwise distribution and Kratky plots showed that it is a multidomain protein with less flexibility when bound to RNA. A 7 Å structure of the complex was obtained through 5000 Cryo-EM micrographs with 41,419 particles that were collected using 200 kV on a JEOL F200 TEM with a K3 direct electron detector and a pixel size of 0.58 Å, magnification 80 K along with a dose of 40 e-/Å2. Significant conformational changes were observed in the protein when bound to RNA and were comparable to P-class proteins. However, RNA is diverting from S-motifs into the editing domain, which could be because the enzyme has edited the RNA. To better understand the path of RNA, data with an inhibitor or an inactive enzyme is required. This study determined that S-class PPR-DYW editing factors have a similar binding mechanism and conformational changes to those of P-class PPR proteins. Upon binding to RNA, the PPR DYW domain interacts with other motifs and appears to have a locked-in conformation. However, the path of RNA and its molecular mechanism are yet to be studied.

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  3. Lenz, H., and V. Knoop. 2013. PREPACT 2.0: Predicting C-to-U and U-to-C RNA Editing in Organelle Genome Sequences with Multiple References and Curated RNA Editing Annotation. Bioinform. Biol. Insights. 7:1–19. doi:10.4137/BBI.S11059.
  4. Okuda, K., A.-L. Chateigner-Boutin, T. Nakamura, E. Delannoy, M. Sugita, F. Myouga, R. Motohashi, K. Shinozaki, I. Small, and T. Shikanai. 2009. Pentatricopeptide repeat proteins with the DYW motif have distinct molecular functions in RNA editing and RNA cleavage in Arabidopsis chloroplasts. Plant Cell. 21:146–156. doi:10.1105/tpc.108.064667.