Structure-functional studies of protein P4 from bacteriophage φ8

Barbora Kascakova¹, Zdenek Franta¹,Tatyana Prudnikova^1,2, Michal Kuty^1,2, Roman Tuma^1,3 and Ivana Kuta Smatanova^1,2

¹University of South Bohemia, Faculty of Science, Branisovska 31, CZ-37005 Ceske Budejovice, Czech Republic

²Academy of Sciences of the Czech Republic, Institute of Nanobiology and Structural Biology GCRC, Zamek 136, 373 33 Nove Hrady, Czech Republic

³University of Leeds, School of Chemistry, The Astbury Centre for Structural Molecular Biology, Leeds LS2 9JT, UK

The dsRNA bacteriophages of Cystoviridae family package their genome into empty capsid – procapsid, which protects the genome from degradation inside as well as outside host cell. The genome packaging is performed by a molecular motor - P4 proteins, which are components of procapsid. The P4s possess an NTPase activity that converts the chemical energy from ATP hydrolysis to a mechanical movement of packaging ssRNA precursors into a procapsid, where the replication and transcription of dsRNA occurs [1- 3]. The P4s are RNA helicases belonging to the Superfamily 4 of helicases with characteristic presence of conserved sequence motifs (H1, H1a, H2, H3 and H4) [1- 3]. The RNA helicases cause the distribution of RNA-protein complexes and carry out RNA unwinding [2]. The P4 assembles into hexameric ring (Fig.1), which has on the outer perimeter NTP-binding sites and the nucleic acid binding sites are located in the central channel. Each P4monomer can be divided into N-terminal , core NTPase domain with sequence motif and C-terminal domain, which is inserted into the central channel of hexamer and its conformational changes regulate ring stability and ATPase activity of P4s [1, 3-4]. Here we grow the monocrystals of the φ8 P4 protein (Fig. 2) in the crystallization conditions of 100mM sodium acetate (pH 4,6) and 2,2 M ammonium sulphate. These conditions were suitable enough to be a good starting point for the next crystallization experiments with RNA assembled to P4.

The work is supported from European Regional Development Fund-Project "Mechanisms and dynamics of macromolecular complexes: from single molecules to cells" (No. CZ.02.1.01/0.0/0.0/15_003/0000441).

1. El Omari K., Meier C., Kainov D., Sutton G., Grimes J.M., Poranen M.M., Bamford D.H., Tuma R., Stuart D.I., Mancini E.J., Nucleic Acids Res., 2013, 41(20), 9396-9410

2. Rabhi M., Tuma R., Boudvillain M., RNA Biol., 2010, 7(6), 655-666

3. Kainov D. E., Tuma R., Mancini E. J., Cell Mol Life Sci 2006, 63, 1095-1105

4. Lisal J., Kainov D. E., Lam T. T., Emmett M. R., Wei H., Gottlieb P., Marshall A. G., Tuma R., Virology 2006, 351 (1), 73-79

Fig. 1. The tertiary structure of P4 hexamer from phage φ8. The monomers are distinguished by different colors [Data from PDB: http://www.rcsb.org/3d-view/4BLQ/1].

Fig. 2. Crystals of protein P4 [5,5mg/ml] from condition with 100 mM sodium acetate (pH 4,6) and 2,2 M ammonium sulphate
(our unpublished data)