A Conserved Tripeptide Sequence at the C-terminus of the Poxvirus DNA Processivity Factor D4 is Essential for Protein Integrity and Function [Microbiology]

November 11th, 2016 by Nuth, M., Guan, H., Ricciardi, R. P.

Vaccinia virus (VACV) is a poxvirus member, and the VACV D4 protein serves both as a uracil-DNA glycosylase (UDG) and as an essential component required for processive DNA synthesis. The VACV A20 protein has no known catalytic function itself, but associates with D4 to form the D4-A20 heterodimer that functions as the poxvirus DNA processivity factor. The heterodimer enables the DNA polymerase to efficiently synthesize extended strands of DNA. Upon characterizing the interaction between D4 and A20, we observed that the C-terminus of D4 is susceptible to perturbation. Further analysis demonstrated that a conserved hexapeptide stretch at the extreme C-terminus of D4 is essential for maintaining protein integrity, as assessed by its requirement for the production of soluble recombinant protein that is functional in processive DNA synthesis. From the known crystal structures of D4, the C-terminal hexapeptide is shown to make intramolecular contact with residues spanning the protein's inner core. Our mutational analysis revealed that a tripeptide motif (215-GFI-217) within the hexapeptide comprises apparent residues necessary for the contact. Prediction of protein disorder identified the hexapeptide and several regions upstream of Gly-215 that comprise residues of the interface surfaces of the D4-A20 heterodimer. Our study suggests that 215-GFI-217 anchors these potentially dynamic upstream regions of the protein in order to maintain protein integrity. Unlike UDGs from diverse sources, where the C-termini are disordered and do not form comparable intramolecular contacts, this feature may be unique to orthopoxviruses.
  • Posted in Journal of Biological Chemistry, Publications
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