| Bacteriophages(phages)are viruses that infect bacteria and represent a simple viral model of basic research with many possibilities for practical application.For example,their potential in the treatment of bacterial infection has been examined since their discovery.The basic research on molecular events and their functional mechanisms in the life cycle of pathogenic bacteriophages will contribute to the development of bacteriophages towards practical applications.phiYY,a dsRNA bacteriophage with a tri-segmented dsRNA genome,was the only member of Cystoviruses found to infect Pseudomonas aeruginosa,a human pathogen.The genome packaging is an important molecular event in the life cycle of phiYY,and the phiYY P4 protein is a genome packaging motor anchored on the viral precursor capsid.P4 converts the chemical energy produced by ATP hydrolysis into mechanical energy to sequentially package the three segments of viral mRNA in the cytoplasm into the precursor capsid.It was reported that P4 proteins from phi6,phi8,phi 12 and phi 13 of Cystoviruses have diversity in structure and biochemical characteristics.Therefore,studying the structure and biochemical characteristics of phiYY P4 has biological significance.In this paper,four crystal structures of phiYY P4 were determined by X-ray crystallography,which are P4wt in apo-form,P4wt combined with tartrate,P4wt combined with PPi-Mg2+,and P41-291 combined with tartrate.Structural analysis showed that the H1 motif exhibits opposite orientation and extended conformation in the tartrate-bound or pyrophosphate-bound structure compared with the apo-form,which may represent an inherent molecular mechanism.Truncation and mutation studies showed that P41-291,P41-300,P41-310,P4E167A,P4N249A,and P4R285A/R290A proteins were mainly hexamer in solution,which was similar to P4wt,indicating that the truncation and mutation did not affect the hexamerization of P4.The NTPase activity results showed that the NTPase activity of P4wt was stimulated by manganese ions and RNA,while the ATPase activities of P4E167A,P4N249A and P4R285A/R290A were significantly reduced,indicating that these residues were all essential for the ATPase activity of P4.It was also found that the deletion of 50 residues at the C terminal of P4 did not damage its ATPase activity.In conclusion,we determined the crystal structures of phiYY P4 protein and detected its biochemical characterisitics.These studies enhanced the understanding of Cystoviruses P4,and provided a molecular basis for further study on the mechanism of ATP hydrolysis of P4 and the genome packaging of phiYY. |
