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Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28

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 describ...

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Autores principales: Reyes-Aldrete, Emilio, Dill, Erik A., Bussetta, Cecile, Szymanski, Michal R., Diemer, Geoffrey, Maindola, Priyank, White, Mark A., Bujalowski, Wlodzimierz M., Choi, Kyung H., Morais, Marc C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823558/
https://www.ncbi.nlm.nih.gov/pubmed/33374840
http://dx.doi.org/10.3390/v13010015
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author Reyes-Aldrete, Emilio
Dill, Erik A.
Bussetta, Cecile
Szymanski, Michal R.
Diemer, Geoffrey
Maindola, Priyank
White, Mark A.
Bujalowski, Wlodzimierz M.
Choi, Kyung H.
Morais, Marc C.
author_facet Reyes-Aldrete, Emilio
Dill, Erik A.
Bussetta, Cecile
Szymanski, Michal R.
Diemer, Geoffrey
Maindola, Priyank
White, Mark A.
Bujalowski, Wlodzimierz M.
Choi, Kyung H.
Morais, Marc C.
author_sort Reyes-Aldrete, Emilio
collection PubMed
description 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 double-stranded 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.
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spelling pubmed-78235582021-01-24 Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28 Reyes-Aldrete, Emilio Dill, Erik A. Bussetta, Cecile Szymanski, Michal R. Diemer, Geoffrey Maindola, Priyank White, Mark A. Bujalowski, Wlodzimierz M. Choi, Kyung H. Morais, Marc C. Viruses Article 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 double-stranded 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. MDPI 2020-12-23 /pmc/articles/PMC7823558/ /pubmed/33374840 http://dx.doi.org/10.3390/v13010015 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Reyes-Aldrete, Emilio
Dill, Erik A.
Bussetta, Cecile
Szymanski, Michal R.
Diemer, Geoffrey
Maindola, Priyank
White, Mark A.
Bujalowski, Wlodzimierz M.
Choi, Kyung H.
Morais, Marc C.
Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28
title Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28
title_full Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28
title_fullStr Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28
title_full_unstemmed Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28
title_short Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28
title_sort biochemical and biophysical characterization of the dsdna packaging motor from the lactococcus lactis bacteriophage asccphi28
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823558/
https://www.ncbi.nlm.nih.gov/pubmed/33374840
http://dx.doi.org/10.3390/v13010015
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