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Sequence and Biochemical Analysis of Vaccinia Virus A32 Protein: Implications for In Vitro Stability and Coiled-Coil Motif Mediated Regulation of the DNA-Dependent ATPase Activity
Version 1
: Received: 2 February 2024 / Approved: 5 February 2024 / Online: 5 February 2024 (07:48:18 CET)
How to cite:
Ramakrishnan, U.; Aggarwal, T.; Kondabagil, K. Sequence and Biochemical Analysis of Vaccinia Virus A32 Protein: Implications for In Vitro Stability and Coiled-Coil Motif Mediated Regulation of the DNA-Dependent ATPase Activity. Preprints2024, 2024020223. https://doi.org/10.20944/preprints202402.0223.v1
Ramakrishnan, U.; Aggarwal, T.; Kondabagil, K. Sequence and Biochemical Analysis of Vaccinia Virus A32 Protein: Implications for In Vitro Stability and Coiled-Coil Motif Mediated Regulation of the DNA-Dependent ATPase Activity. Preprints 2024, 2024020223. https://doi.org/10.20944/preprints202402.0223.v1
Ramakrishnan, U.; Aggarwal, T.; Kondabagil, K. Sequence and Biochemical Analysis of Vaccinia Virus A32 Protein: Implications for In Vitro Stability and Coiled-Coil Motif Mediated Regulation of the DNA-Dependent ATPase Activity. Preprints2024, 2024020223. https://doi.org/10.20944/preprints202402.0223.v1
APA Style
Ramakrishnan, U., Aggarwal, T., & Kondabagil, K. (2024). Sequence and Biochemical Analysis of Vaccinia Virus A32 Protein: Implications for In Vitro Stability and Coiled-Coil Motif Mediated Regulation of the DNA-Dependent ATPase Activity. Preprints. https://doi.org/10.20944/preprints202402.0223.v1
Chicago/Turabian Style
Ramakrishnan, U., Tanvi Aggarwal and Kiran Kondabagil. 2024 "Sequence and Biochemical Analysis of Vaccinia Virus A32 Protein: Implications for In Vitro Stability and Coiled-Coil Motif Mediated Regulation of the DNA-Dependent ATPase Activity" Preprints. https://doi.org/10.20944/preprints202402.0223.v1
Abstract
Nucleocytoplasmic large DNA viruses (NCLDVs) have massive genomes and particle sizes compared to other known viruses. NCLDVs, including poxviruses, encode ATPases of the FtsK/HerA superfamily to facilitate genome encapsidation. However, their biochemical and structural characteristics are yet to be discerned. In this study, we demonstrate that the viral ATPases are significantly shorter than their bacterial homologs, representing the minimal ATPase core of the FtsK/HerA superfamily. We analyzed the sequence and structural features of the vaccinia virus A32 protein and determined their roles in the stability of the A32 protein and its ATPase activity. We sought to purify A32 by various techniques and noted that recombinant A32 expressed in E. coli is highly insoluble and unstable in solution, possibly because of the disordered C-terminus. N-terminal fusion with the thioredoxin solubility tag could alleviate this issue to some extent, but subsequent tag cleavage results in increased susceptibility to precipitation and degradation. We also predicted a conserved coiled-coil motif (CCM) towards the C-terminus of VV A32. ATPase activity of A32 is known to increase in the presence of DNA. Our comparative analysis of wildtype A32 versus CCM mutants suggests that this DNA dependence of A32’s ATPase activity is likely regulated by the CCM. Since CCM is also known to facilitate protein oligomerization, these findings provide new opportunities for further detailed characterization of A32.
Keywords
Genome packaging ATPase; vaccinia virus; coiled coil motif; FtsK/HerA superfamily; Nucleo-cytoplasmic large DNA viruses
Subject
Biology and Life Sciences, Biochemistry and Molecular Biology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
