A Conserved Tripeptide Sequence at the C Terminus of the Poxvirus DNA Processivity Factor D4 Is Essential for Protein Integrity and Function

Abstract

Vaccinia virus (VACV) is a poxvirus, and the VACV D4 protein serves both as a uracil-DNA glycosylase 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 inner core of the protein. Our mutational analysis revealed that a tripeptide motif (²¹⁵GFI²¹⁷) within the hexapeptide comprises apparent residues necessary for the contact. Prediction of protein disorder identified the hexapeptide and several regions upstream of Gly²¹⁵ that comprise residues of the interface surfaces of the D4-A20 heterodimer. Our study suggests that ²¹⁵GFI²¹⁷ anchors these potentially dynamic upstream regions of the protein to maintain protein integrity. Unlike uracil-DNA glycosylases from diverse sources, where the C termini are disordered and do not form comparable intramolecular contacts, this feature may be unique to orthopoxviruses.

Keywords: DNA viruses; NMR; poxvirus; processivity; protein-protein interaction; surface plasmon resonance (SPR); viral replication.

FIGURE 1.

Recombinant protein expression of A20. A, Coomassie staining of the purified proteins used in the study resolved on a 4–12% BisTris gel. Because both of the N-terminal (N-term) and C-terminal His-tagged proteins of D4 could be purified/enriched by Ni-NTA as well as detected by anti-His antibody during SPR experiments and Western blotting (WB), it indicates that the observed doublet bands do not depict potential protein truncation because of early terminated translation or damage during purification. Therefore, the observed doublets for all three D4 versions could likely be due to varying degrees of SDS binding during electrophoresis. aa, amino acids. B, bacterial expression of the N-terminal regions of A20 in the absence of the MBP carrier protein. The crude lysate contains both the insoluble and soluble fractions, whereas the cleared lysate, containing only the soluble fraction, was obtained after sedimentation of the crude lysate by 10-min centrifugation at 15,000 rpm. Only the crude preparation is shown for the uninduced cells (U). Based on the predicted molecular weights (as N-terminal His-tagged constructs), the respective proteins are shown boxed. Additionally, when the pellets were collected after sedimentation, the truncated proteins were readily solubilized with 6 m guanidine HCl (data not shown). C, expression of the N-terminal 100 amino acids in baculovirus and detection with anti-His antibody (arrowhead).

FIGURE 2.

A and B, SPR binding profiles of MBP-A20_1–63 to captured His₆D4 (A) and D4His₆ (B). The injection of 20 μm purified MBP is shown (dashed line). The measurements of 10 (light gray) and 20 μm (dark gray) MBP-A20_1–63 were excluded from curve fitting because of high data dispersion. The sensogram overlays reflect the mean fit values (black solid lines) ± S.D. (gray) of three repeats from the injections of 0.125–5 μm MBP-A20_1–63 (bottom to top). RU, response units. C, the temperature dependence of binding to MBP-A20_1–63 as depicted by linear (dashed lines; R² = 0.31 and 0.54 for His₆D4 and D4His₆, respectively) and nonlinear (solid lines; R² = 0.72 and 0.91 for His₆D4 and D4His₆, respectively) fit. Shown are the mean ± S.D. of three repeats. D, summary of the kinetic data determined from A and B, with example rates used for the nonlinear fit shown. E, extracted thermodynamic parameters determined from C, where ΔG, ΔH, and ΔS values were obtained by the linear fit and ΔC_p by the nonlinear fit. Entropy values are shown for 25 °C.

FIGURE 3.

A–D, the impact of the His₆ tag on the structural and stability properties of D4 proteins as determined by far- and near-UV CD spectroscopy (A), ¹H-¹⁵N HSQC NMR spectroscopy (B and C), and steady-state UV-visible spectroscopy (D). D, the values for the soluble fractions were determined relative to untreated proteins. All values reflect the mean ± S.D. of three repeats. The p values were determined by analysis of variance in comparison with the untagged D4 for the corresponding temperature treatments. *, p < 0.001; rt, room temperature; MRW, mean residual weight.

FIGURE 4.

Protein expression and function of the deletion mutants of the C-terminal hexapeptide stretch. A, construct designs of the D4 deletion mutants. B, proteins from bacterial expression were resolved and detected as described in Fig. 1, where C refers to the crude lysate, S the soluble fraction, and U the crude lysate of the uninduced cells. WB, Western blot. C, in vitro DNA synthesis activity was measured using proteins produced by the cell-free method. The corresponding D4 protein levels are shown by autoradiography of [³⁵S]cysteine incorporation. To permit sample order, the image of the WT is a splice of the same gel. The levels of DNA synthesis activity reflect the mean ± S.D. of three to six repeats, with the indicated p value in comparison with the WT.

FIGURE 5.

A–D, protein expression and function of the C-terminal swaps (A and C) and alanine mutants (B and D). Conditions and notations are the same as in Fig. 4. C and D, all values reflect the mean ± S.D. of three repeats, with the p values shown in comparison with the WT. D, the autoradiographic image of the WT is a splice of the same gel to depict sample order. WB, Western blot; S, soluble fraction; C, crude lysate; U, crude lysate of uninduced cells; Ec, E. coli; Hs, H. sapiens.

FIGURE 6.

Properties of the C-terminal region of the D4 protein. A, protein sequence alignment of the C terminus of VACV D4 to representative UDGs. Shown are representatives from orthopoxviruses (CMLV, camelpox; CPX, cowpox; ECTV, mousepox; HSPV, horsepox; MPV, monkeypox; RCNV, raccoonpox; TPV, tanapox; VARV; and YKPV, Yoka poxvirus), cervidpoxvirus (DPV, deerpox), avipoxvirus (FWPV, fowlpox), capripoxvirus (GTPV, goatpox), molluscipoxvirus (MCV), leporipoxvirus (MXV, myxoma virus), parapoxvirus (ORFV, orf virus), suipoxvirus (SWPV, swinepox), yatapoxvirus (YLD, Yaba-like disease virus), and UDGs with reported crystal structures (Ec, E. coli, PDB code 2EUG; Hs, H. sapiens, PDB code 1AKZ; and HSV type 1, PDB code 1LAU). Of note, only VACV is structurally available among the orthopoxviruses. The maroon solid line marks the hexapeptide stretch. B, protein solubility summary of single amino acid substitutions at the hexapeptide stretch. Proteins from bacterial expressions that were undetectable by Western blotting are classified as insoluble (red), whereas detections in both the crude and soluble fractions are identified as soluble (green). Proteins detected with a minor presence in the soluble fraction are identified as in between (orange). Efforts were made to choose substitutions that would alter the physical and/or chemical properties at the sites of interest. Therefore, mutations that either dramatically interfered with protein solubility with only minor changes (e.g. the Gly²¹⁵ site) or produced no effects with significant substitutions (e.g. the Gln²¹⁴ site) were pursued only sparingly. Unpursued mutations are shown uncolored. The asterisk indicates that the assessment is from the C-terminal swap of MCV, which contains Val²¹⁷ in addition to ²¹⁸PL²¹⁹ (Fig. 5A).

FIGURE 7.

Protein disorder and the role of ²¹⁵GFI²¹⁷. A, the diagram depicts D4 as a monomer (PDB code 2OWR), with the hexapeptide shown as a green ribbon and the side chains of ²¹⁵GFI²¹⁷ as orange sticks. The hexapeptide sequence is shown with the corresponding color scheme. In the crystal structure, residues spanning the region 102–113 (magenta) are in close proximity to ²¹⁵GFI²¹⁷ and are presumably responsible for the bulk of the intramolecular contact. B, the five regions of disorder predicted by DisEMBL. C, a proposed model depicting the role of ²¹⁵GFI²¹⁷ in maintaining protein integrity and, thus, function. Through intramolecular interaction, ²¹⁵GFI²¹⁷ (shown with a similar color scheme as in A) anchors the potentially dynamic regions of D4 (with only the predicted region 201–218 shown highlighted in red) that are important for target recognition (e.g. A20 and DNA). The removal of ²¹⁵GFI²¹⁷ or the weakening of the intramolecular contact by amino acid substitutions untethers the dynamic region of D4 and is an energetic liability. As a result, protein misfolding ensues, leading to protein heterogeneity and aggregation and, ultimately, protein precipitation. Because of the hydrophobic nature of the protein interface, it is presumed that the binding surfaces of D4 will be buried within these aggregates. In this scenario, A20 or DNA is incapable of binding to soluble aggregated D4 proteins because of the lack of access to the binding surfaces. As an explanation for the observed instability exhibited by D4 in solution, the promotion of protein aggregation and heterogeneity is likely rooted in dynamics still present for the homodimer.