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Project Number18-74-00123

Project titleStudy of DNA-protein interactions in terminal complexes of bacteriophages infecting thermophilic bacteria

Project LeadKlimuk Evgeny

AffiliationAutonomous Non-Profit Organization for Higher Education "Skolkovo Institute of Science and Technology",

Implementation period2018 - 2019

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-202 - Proteomics; structure and functions of proteins

KeywordsViruses, bacteriophages, thermophilic bacteria, DNA packaging motor, DNA-protein complexes, small terminase, X-ray analysis, 3D structures



Viruses are the most abundant biological agents on our planet, able to infect organisms of all domains of life. The packaging of viral genome into a capsid is essential part of viral life cycle. Some viruses employ the particle–genome coassembly mechanism, while others specifically package their genome into a preformed capsid by specialized molecular machines fuelled by energy derived from macroergic compounds hydrolysis. While there are structural and functional similarities, these machines have evolved to perform different tasks and many of them are still poorly characterised. DNA packaging motors used by bacteriophages, or viruses of bacteria, are the most studied to date. Tailed bacteriophages and evolutionarily-related pathogenic herpesviruses (Epstein-Barr, Cytomegalovirus, Varicella Zoster) encode a DNA packaging motor that contains 3 proteins: portal protein, which forms the gateway for the entry of DNA into the capsid, as well as the i) small and ii) large subunits of the terminase, responsible for i) recognition of phage DNA and ii) its translocation using energy released during ATP hydrolysis followed by the cleavage of the concatemeric genomic DNA to package one full-genomic equivalent. Non-tailed bacteriophages and evolutionarily-related mammalian pathogens (such as poxviruses), also translocates DNA into pre-formed capsids using an ATP-driven motor localised at a single vertex. Unlike DNA packaging motors of tailed bacteriophages, this group of phages does not encode a portal protein, and instead utilises a virion-attached ATPase with 12-fold symmetry. Covalently 5’-end linked terminal proteins are necessary for genome packaging initiation and termination. Though in recent years progress in understanding the 3D structures of individual components of DNA packaging motors of tailed bacteriophages was achieved and some functional data generated using innovative “single-molecule" experiments were obtained, the mechanism of DNA packaging into viral particles is still unknown. Specifically, there is still no understanding of the mechanisms of DNA recognition and initiation of packaging process. For non-tailed bacteriophages, even structural information on individual components of DNA packaging motors is extremely poor. During this project, we are going to obtain structural data for the components of the terminal complex of DNA packaging motors, as well as characterize the DNA-protein complexes formed by these terminal proteins on the model of bacteriophages infecting thermophilic bacterium Thermus thermophilus. Several such bacteriophages were recently isolated and characterized in our laboratory. It is known that proteins from thermophilic organisms are more stable and compact, making it easier to obtain high quality crystals. As results of preliminary experiments we and our collaborators obtained diffracting crystals for individual components of DNA-packaging motors from several such phages and resolved their 3D structures. For example, we resolved 3D structures for portal protein and nuclease domain of the large subunit of the terminase encoded by tailed bacteriophage G20c. We aim to use the structural, biophysical and biochemical approaches to elucidate the molecular basis of DNA recognition and initiation of DNA packaging by the terminal complex of DNA packaging motors encoded by bacteriophages infecting thermophilic bacterium Thermus thermophilus.

Expected results
As part of the project, structural and functional characterization of the components of the terminal complex participating in DNA packaging as well as DNA-protein complexes formed by them from the bacteriophages G18 and phiKo, infecting the thermophilic bacterium Thermus thermophilus, will be carried out. These bacteriophages were recently isolated in our laboratory from water samples collected from hot springs in Kamchatka, the sequence of its genomic DNA was completely determined and functionally annotated during preliminary studies. Small subunit of terminase is responsible for recognition of phage DNA for packaging. These proteins are highly divergent with no recognizable amino acid sequence similarity for proteins from different phages. Nevertheless it is known that usually small and large terminase subunits, and the portal protein genes form an operon in phage genomes. In such operons, gene that encodes small subunit of terminase is usually expressed first. Predicted small terminase subunit of the bacteriophage G18 obtained by heterologous expression in E. coli cells forms a 9-subunit oligomer. Such oligomeric state is typical for proteins of this class. We aim to determine 3D structure of the small subunit of terminase encoded by the bacteriophage G18. In addition, pac site (packaging initiation site) in the bacteriophage G18 genome will be determined experimentally. Finally, we aim to elucidate the structure and mechanism of small terminase / pac site complex formation. Structural data on the components of the DNA packaging motor of non-tailed bacteriophages is extremely poor. Recent asymmetric cryo-EM reconstruction of non-tailed bacteriophage PRD1 capsid (~12 Å resolution) revealed a motor assembly exhibiting 12-fold symmetry. Limited solubility and stability of the PRD1 FtsK-like ATPase (P9 protein) and other proteins involved in DNA packaging, e.g., the terminal protein P8, does not allow to crystallize them and solve their 3D-structures. Indeed the only high-resolution structural information available for components of the DNA packaging motor of non-tailed viruses relates to FtsK-like ATPase, protein B204, from archaeal virus STIV2, that is distant homologue of PRD1 P9 protein (shares only 16% sequence identity). Within the framework of the project, we will produce the FtsK-like motor protein from thermophage phiKo and resolve its 3D structure. In parallel we expect to identify other components of the motor, including the terminal protein, by co-IP. Finally we aim to elucidate the structure and mechanism of terminal protein / DNA complex formation. There is no doubt that results we plan to obtain will be new and original and will be of fundamental importance for the understanding structural and functional organization of the bacteriophage DNA packaging machinery. Furthermore, the expected results will be of utmost importance for biotechnology and medicine, in particular for the targeted delivery of nucleic acids by viral terminal proteins.



Annotation of the results obtained in 2019
During the second year of the project, most of the tasks were completed. The crystallization conditions of small terminase subunit of the bacteriophage G18 and the motor ATPase of the bacteriophage phiKo were selected and their 3D structures were resolved. Alternative experimental models have been created to confirm the packaging initiation site for small terminase subunit of the bacteriophage G18, one of which is based on the close relationship of the bacteriophages G18 and phiFa. For the bacteriophage phiFa, a model for packaging genomic DNA in a capsid is proposed. Confirmation of this model made it possible to reconsider the organization of its genome, which in turn explained the features of the adaptation process of CRISPR type III systems observed in experiments. The results of this work are published. The expression profile of the motor ATPase of the bacteriophage phiKo during infection was characterized, and experiments on co-immunoprecipitation of proteins associated with it were carried out. The results are presented at the FEMS Congress 2019 conference.



1. Артамонова Дарья, Карнеева Карина, Медведева Софья, Климук Евгений, Колесник Матвей, Северинов Константин Spacer acquisition by Type III CRISPR-Cas system during bacteriophage infection of Thermus thermophilus Nucleic Acids Research, - (year - 2020).

Annotation of the results obtained in 2018
During the first year of the project, the most routine part of the work was carried out. Constructs for expression of the small terminase subunit of bacteriophage G18 and the motor ATPase of bacteriophage phiKo as fusions with N-terminal 6 * His-tag and N-terminal SUMO-tag were created. Conditions for the expression and purification of these proteins were selected and samples of proteins with a purity of > 99% were obtained. Crystallization conditions were tested for these proteins and 6 crystals with small terminase subunit of bacteriophage G18 were sent to Diamond synchrotron for data collection. A packaging initiation site was determined for small terminase subunit of bacteriophage G18 using the CHIP-seq method. In addition, using newly developed PhageTerm algorithm, the end of genomic DNA of bacteriophage G18 was determined, it corresponds to position 27296. Finally, highly specific antibodies were obtained for the phiKo bacteriophage motor ATPase, these antibodies will be used to co-immunoprecipitate components of the terminal complex of bacteriophage phiKo.