NMR structure of a vestigial nuclease provides insight into the evolution of functional transitions in viral dsDNA packaging motors.

Erik A Dill,Kyung H Choi,Bryon P Mahler,Paul J Bujalowski,Marc C Morais,Huzhang Mao,Paul J Jardine

doi:10.1093/nar/gkaa874

Abstract

Double-stranded DNA viruses use ATP-powered molecular motors to package their genomic DNA. To ensure efficient genome encapsidation, these motors regulate functional transitions between initiation, translocation, and termination modes. Here, we report structural and biophysical analyses of the C-terminal domain of the bacteriophage phi29 ATPase (CTD) that suggest a structural basis for these functional transitions. Sedimentation experiments show that the inter-domain linker in the full-length protein promotes oligomerization and thus may play a role in assembly of the functional motor. The NMR solution structure of the CTD indicates it is a vestigial nuclease domain that likely evolved from conserved nuclease domains in phage terminases. Despite the loss of nuclease activity, fluorescence binding assays confirm the CTD retains its DNA binding capabilities and fitting the CTD into cryoEM density of the phi29 motor shows that the CTD directly binds DNA. However, the interacting residues differ from those identified by NMR titration in solution, suggesting that packaging motors undergo conformational changes to transition between initiation, translocation, and termination. Taken together, these results provide insight into the evolution of functional transitions in viral dsDNA packaging motors.

Highlights

A fundamental step in the life cycle of any virus is encapsidation of the viral genome within a protein shell, or capsid, which protects the genome from environmental assault as the virus transits between hosts
Analytical ultracentrifugation, fluorescence-based binding assays, and NMR chemical shift perturbation (CSP) experiments were used to characterize ATPase assembly and nucleic acid binding by the CTD. These results show how the CTD interacts with other motor components and the translocating DNA and provides insight into how viral dsDNA packaging motors transition between genome processing and translocation functions
We observed a constant population of only 2% dimers for the shorter CTD at concentrations of 30 and 85 μM (Fig 3B, C). These results indicate that the N-terminal tail of the construct that included the interdomain linker region (CTD-L) induces dimerization

Summary

Introduction

A fundamental step in the life cycle of any virus is encapsidation of the viral genome within a protein shell, or capsid, which protects the genome from environmental assault as the virus transits between hosts. An empty virus shell is first assembled, and the genome is actively packaged into this pre-formed container This is the strategy used by some ssRNA [1] and ssDNA viruses [2, 3], and virtually all dsDNA viruses such as herpes virus, pox virus, adenovirus, and all the tailed dsDNA bacteriophages [4]. This second strategy is remarkable considering the enthalpic, entropic, and DNA bending energies that must be overcome to package DNA to near crystalline densities within the confined space of the capsid

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Discussion

Conclusion