Biochemical and biophysical characterization of the dsdna packaging motor from the lactococcus lactis bacteriophage asccphi28

Emilio Reyes-Aldrete, Erik A. Dill, Cecile Bussetta, Michal R. Szymanski, Geoffrey Diemer, Priyank Maindola, Mark A. White, Wlodzimierz M. Bujalowski, Kyung H. Choi, Marc C. Morais

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccϕ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other doublestranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

Original languageEnglish (US)
Article number15
JournalViruses
Volume13
Issue number1
DOIs
StatePublished - Jan 2021

Keywords

  • ASCE superfamily
  • ATPase
  • Asccphi28
  • Bacteriophage
  • DsDNA virus
  • Encapsidation protein
  • Genome packaging
  • Molecular motor
  • P-loop ATPase
  • Phage terminase
  • Phi29

ASJC Scopus subject areas

  • Infectious Diseases
  • Virology

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