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Biophysical and Structural Characterization of a Viral Genome Packaging Motor

Like many dsDNA viruses, bacteriophage λ replicates its genome as a concatemer consisting of multiple copies of covalently linked dsDNA genomes. To encapsidate a single genome within a nascent procapsid, λ must: 1) find its own dsDNA amongst the multitude of host nucleic acids; 2) identify the genomic start site; 3) cut the DNA; 4) bring the excised DNA to a procapsid; 5) translocate DNA into the capsid; 6) cut DNA again at a packaging termination site, 7) disengage from the newly filled capsid; and 8) bring the remainder of the genomic concatemer to fill another empty procapsid. These disparate genome processing tasks are carried out by a single virus-encoded enzyme complex called terminase. While it has been shown that λ terminase initially forms a tetrameric complex to cut DNA, it is not clear whether the same configuration translocates DNA. Here, we describe biophysical and initial structural characterization of a λ terminase translocation complex. Analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS) indicate that between 4 and 5 protomeric subunits assemble a cone-shaped terminase complex with a maximum dimension of ∼230 and radius of gyration of ∼72 Å. Two-dimensional classification of cryoEM images of λ terminase are consistent with these dimensions and show that particles assume a preferred orientation in ice. The orientations appear to be end-on, as terminase rings resemble a starfish with approximate pentameric symmetry. While ∼5-fold symmetry is apparent, one of the five “arms” appears partially displaced with weaker more diffuse density in some classes, suggesting flexibility and/or partial occupancy. Charge detection mass spectrometry (CDMS) is consistent with a pentameric complex, with evidence that one motor subunit is weakly bound. Kinetic analysis indicates that the complex hydrolyzes ATP at a rate comparable to the rates of other phage packaging motors. Together with previously published data, these results suggest that λ terminase assembles conformationally and stoichiometrically distinct complexes to carry out different genome processing tasks. We propose a “symmetry resolution” pathway to explain how terminase transitions between these structurally and functionally distinct states.