Caracterización de la terminasa mayor del bacteriófago T7

Resumen

DNA packaging inside preformed proheads takes place in a similar way in tailed bacteriophages as well as in some animal viruses (such as Herpesvirus and Adenovirus). The structural and functional complexity of phages accomplished by its genetic simplicity makes them a good model system to study viral packaging. The morphogenetic pathway in double stranded DNA (dsDNA) phages, except for some individual features, is highly conserved owing to their common evolutionary origin. The assembly begins with the formation of empty proheads composed of the capsid proteins, the scaffolding proteins and the connector (that links the viral head and the tail). DNA is translocated into the preformed prohead by the portal complex, located at a special prohead vertex. The portal is made up two components, the terminase and the connector. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Due to its dual activity, ATPase and nuclease, and transiently interacts with the connector, the terminase is a very unstable protein. Once the DNA is packaged inside the mature head, the terminase dissociates and the tail proteins are incorporated to build the fully infective virion. Here, we have determined the structure of the large terminase (gp19) of phage T7 by electron microscopy and image processing techniques. The pentameric terminase presents a channel wide enough to accommodate dsDNA. The three-dimensional volume of the complete portal complex is also reported, revealing the coupling between the terminase and the connector through a continuous channel. The structure of the terminase assembled into the portal complex showed a different conformation when compared with the isolated terminase pentamer. To understand at molecular level the terminase morphological change and to infer the location of its catalytic motifs, we generated the terminase atomic model based on the crystallographic structure of its phage T4 counterpart. The docking of the threaded model in both terminase conformations showed that the transition between the two states can be achieved without any intra-domain bending. The two terminase conformations may be involved in the sequential DNA translocation, showing eventually two steps of the packaging mechanism. We also used a surface stress nanomechanical sensor to study ATP-induced conformational changes in the T7 terminase. The recording of the cantilever bending during the terminase immobilization upon its surface shows the existence of a gp19 monolayer arrangement confirmed by atomic force microscopy. This two-dimensional arrangement is characteristic of low terminase concentrations (below 40 ng/ml). The ATP hydrolysis of the terminase generates a stepped motion of the cantilever and points to a mechanical cooperative effect among gp19 oligomers. Furthermore, the effect of ATP can be counteracted by non-hydrolyzable nucleotide analogs. Finally, the inherent aggregation tendency of the terminase was examined by raising the concentration assayed. We generate a gp19 multilayer arrangement over the cantilever surface, by immobilizing terminase concentrations up to 4 ¿g/ml. The response of the 3D matrix to the binding of ATP is completely distinct compared with the monolayer of proteins. ATP interaction modifies the conformation of the proteins that build the clusters thus affecting the cohesion of the aggregates. As the gp19 clusters can bind ATP and AMP-PNP (a non hydrolyzable analog) producing a genuine deflection of the microcantilever, the competitive binding between them can be also detected in real time.