Large terminase conformational change induced by connector binding in bacteriophage T7

Abstract

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy. The five terminase subunits assemble in a toroid that encloses a channel wide enough to accommodate dsDNA. The structure of the complete connector-terminase complex is also reported, revealing the coupling between the terminase and the connector forming a continuous channel. The structure of the terminase assembled into the complex showed a different conformation when compared with the isolated terminase pentamer. To understand in molecular terms the terminase morphological change, 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 by rigid body subunit rotation in the pentameric assembly. The existence of two terminase conformations and its possible relation to the sequential DNA translocation may shed light into the molecular bases of the packaging mechanism of bacteriophage T7.

Keywords: ATPases; Bacteriophage; DNA Packaging; DNA Translocation; Electron Microscopy (EM); Protein Conformation; Single Particle Reconstruction; Terminase; Virus Assembly.

FIGURE 1.

Purification of the oligomeric active terminase and EM analysis. A, SDS-polyacrylamide gel electrophoresis of the fractions from a GraFix centrifugation show the presence of the gp19 monomer (fractions 3–8) and oligomer (box, fractions 5–11). Bands were detected by staining with Coomassie Brilliant Blue. L, load; Std, protein standards; lane B, bottom of the centrifuge tube. B, shown is SDS-polyacrylamide gel electrophoresis of the fractions from a gradient centrifugation of a mixture of molecular mass markers: bovine serum albumin (66 kDa, peak in fraction 3–5), aldolase (158 kDa, peak in fraction 5–9) and ferritin (440 kDa, peak in fraction 11–13). Lane B, bottom of the centrifuge tube. C, shown is measurement of the gp19 ATPase activity. ATPase assays were tested in duplicate; − represents buffer control. The concentration of gp19 tested in each assay was 0.45 μm. The box highlights the active oligomeric fractions from 5 to 9. Pi represents inorganic phosphate. D shows a negatively stained sample of a field of purified large terminases (arrows). Lower row, two-dimensional averaged images of the oligomeric terminase. E, rotational analysis of the harmonic components (x axis) of the end-on averaged image of the terminase shows the existence of 5-fold symmetry (percentage of total rotational power in the y axis). Inset, averaged image with imposed pentameric symmetry. The scale bar represents 100 Å.

FIGURE 2.

Three-dimensional reconstruction of the pentameric large terminase and fitting of the gp19 atomic model. A, shown is an EM reconstruction of the pentameric terminase. The structure is shown in three different orientations, as indicated. B, sequence alignment used for the modeling of the T7 large terminase (gp19, target protein) based on the template structure of the T4 large terminase (gp17, 3CPE) shows conserved motifs at the amino acid level and the secondary structures correspondence. C, shown is a comparison of the gp17 atomic structure (in orange) and the gp19 final model (in blue). D, shown is a translucent model of the terminase structure together with the fitted atomic model of the gp19 pentamer in end-on and side views. Each gp19 monomer is presented in a different color. Inset, two detailed views of the fitted monomer in different orientations. The scale bar represents 50 Å.

FIGURE 3.

Purification and characterization of the connector-terminase complex. A, shown is an electrophoretic analysis of the fractions from glycerol gradient centrifugation of purified terminase (upper panel), connector (middle panel), and connector-terminase complex (lower panel). L, load; B, bottom of the gradient. The gp19 monomer was concentrated in fractions 3–5 (box), the peak of oligomeric connectors corresponded to fractions 7–9 (box), and an estequiometric proportion of both oligomeric proteins was observed in fractions 11–12 of the lower panel gradient (box), suggesting the existence of a putative complex. B, shown are projection images from the three-dimensional reconstructed model (upper row) and averaged views from the experimental images (lower row). The arrowhead points the proposed interface between the terminase and the connector assemblies. The scale bar corresponds to 100 Å. C, side, end-on, and bottom views of the three-dimensional reconstruction of the complex show the two morphologically different domains. The longitudinal axis of the complex is 220 Å, and the maximum diameter is 190 Å. The scale bar represents 50 Å.

FIGURE 4.

Topology of the connector-terminase complex and docking of the gp19 atomic model. A, shown is filtered reconstruction of the cryo-EM model of the T7 connector. B, shown is three-dimensional reconstruction of the connector by negative staining. C, the three-dimensional volume of the connector (in cyan) fits well in the upper domain of the connector-terminase complex (in gray mesh). A schematic of the position of the complex in the procapsid is also included (in orange and yellow). D, shown is difference volume (in red) between the complex and the connector; besides some minor differences in the crown and wings of the connector, the main difference volume corresponds to the lower domain of the complex, which has been assigned to the terminase. E, shown is a surface representation of the pentameric terminase segmented from the connector-terminase complex in two different orientations, as indicated. F, shown is fit of the gp19 atomic model into the segmented volume of the terminase complexed with the connector. Two detailed views of monomer docking are shown in distinct orientations as indicated (left) and the docking of the pentameric atomic model in end-on view (right). G, location of the catalytic regions of the terminase structure in the connector-bound conformation is shown. ATPase motifs are shown in red, and the nuclease region is in cyan. The scale bars represent 50 Å.

FIGURE 5.

Two distinct conformations of the terminase pentamer; isolated terminase and complexed after interaction with the connector. A, shown is fit of the gp19 monomer atomic model into the EM envelopes of the isolated (yellow) and complex-segmented terminase (gray). B, shown is rigid body transition of the gp19 monomer atomic model from the isolated (upper site) to the complex conformation (lower site). Each helix is depicted in a different color to highlight the global movement and the absence of intramolecular bending. C, shown is electrostatic potential of the isolated terminase surface in end-on view (amino domain up) and side section of the internal channel (lower panel). D, surface potential of the complex-segmented terminase in an equivalent end-on view (upper panel) and cross-section of the internal channel (lower panel) are shown. The scale bar represents 50 Å.