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. 2017 Oct 20;9(10):305.
doi: 10.3390/v9100305.

Structural and Functional Insight into Canarypox Virus CNP058 Mediated Regulation of Apoptosis

Mohd Ishtiaq Anasir  1 , Amy A Baxter  2 , Ivan K H Poon  3 , Mark D Hulett  4 , Marc Kvansakul  5
Affiliations

Structural and Functional Insight into Canarypox Virus CNP058 Mediated Regulation of Apoptosis

Mohd Ishtiaq Anasir et al. Viruses. .

Abstract

Programmed cell death or apoptosis is an important component of host defense systems against viral infection. The B-cell lymphoma 2 (Bcl-2) proteins family is the main arbiter of mitochondrially mediated apoptosis, and viruses have evolved sequence and structural mimics of Bcl-2 to subvert premature host cell apoptosis in response to viral infection. The sequencing of the canarypox virus genome identified a putative pro-survival Bcl-2 protein, CNP058. However, a role in apoptosis inhibition for CNP058 has not been identified to date. Here, we report that CNP058 is able to bind several host cell pro-death Bcl-2 proteins, including Bak and Bax, as well as several BH3 only-proteins including Bim, Bid, Bmf, Noxa, Puma, and Hrk with high to moderate affinities. We then defined the structural basis for CNP058 binding to pro-death Bcl-2 proteins by determining the crystal structure of CNP058 bound to Bim BH3. CNP058 adopts the conserved Bcl-2 like fold observed in cellular pro-survival Bcl-2 proteins, and utilizes the canonical ligand binding groove to bind Bim BH3. We then demonstrate that CNP058 is a potent inhibitor of ultraviolet (UV) induced apoptosis in a cell culture model. Our findings suggest that CNP058 is a potent inhibitor of apoptosis that is able to bind to BH3 domain peptides from a broad range of pro-death Bcl-2 proteins, and may play a key role in countering premature host apoptosis.

Keywords: Bcl-2; X-ray crystallography; apoptosis; avipoxvirus; isothermal titration calorimetry; poxvirus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
CNP058 is a monomer in solution. Size exclusion chromatography of CNP058 using a Superdex 200 Increase 3.2/300 column. The elution volume of the peak of interest (CNP058) is 1.77 mL (solid line). The molecular weight standards shown are albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome c (Cyt c) (12 kDa) (all source from Sigma Aldrich), shown as dotted lines and labeled on top of the respective peaks. AU: absorbance units.
Figure 2
Figure 2
CNP058 interacts with Bak and Bax as well as all BH3-only proteins except Bad and Bik. Raw heats measured by isothermal titration calorimetry (ITC) for CNP058 interactions with Bcl-2 homology domain 3 (BH3) domain peptides of pro-death B-cell lymphoma 2 (Bcl-2) proteins from Homo sapiens (HS) and Gallus gallus (GG).
Figure 3
Figure 3
Structure based sequence alignment of CNP058 with FPV039 and cellular Bcl-2 protein Mcl-1. The α-helical secondary structure elements indicated (denoted as H and labeled as Helix 1–8) are based on the crystal structure of CNP058. Bcl-2 homology domains (BH domains) 1–4 are marked underneath the aligned sequences. Conserved residues are highlighted in yellow. “*” denotes conserved residues for all three proteins, “:” denotes highly similar residues between the three proteins, and “.” denotes weakly similar residues between the three proteins. The NWGR or TWGR motifs are marked in bold. Uniprot accession codes: CNP058 = Q6VZT9, FPV039 = Q9J5G4 and Mcl-1 = Q07820; PDB accession codes: FPV039 = 5TZP and Mcl-1 = 2NL9.
Figure 4
Figure 4
2Fo-Fc electron density maps of CNP058:Bim BH3 domain complex. Electron density map encompassing the hydrophobic binding groove of CNP058 in complex with Bim BH3. CNP058 is shown as yellow sticks, whereas Bim BH3 is shown as green sticks. The electron density map is shown as a blue mesh contoured at 1 δ.
Figure 5
Figure 5
CNP058 interacts with pro-death Bcl-2 proteins in a BH3-domain hydrophobic groove dependent manner. (A) Cartoon representation of CNP058:Bim BH3 domain complex, (B) FPV039:Bik BH3 domain complex and (C) Mcl-1:Bim BH3 domain complex. The view is into the conserved hydrophobic ligand binding groove formed by α-helices 2–5. PDB ID: FPV039:Bik = 5TZP, Mcl-1:Bim = 2NL9. (D) CNP058:Bim complex (yellow) was superimposed onto FPV039:Bik complex (cyan). Bim and Bmf were removed from respective complex structures for clarity.
Figure 6
Figure 6
CNP058:Bim BH3 complex interface. Surface representation of (A) CNP058:Bim BH3 domain complex, (B) FPV039:Bik BH3 domain complex, and (C) Mcl-1:Bim BH3 domain complex. The view is into the conserved hydrophobic ligand binding groove formed by α-helices 2–5. The surfaces are shown in grey. Residues involved in interactions are shown as sticks and labeled. Hydrogen bonds and ionic interactions between CNP058 and Bim are denoted as black dotted lines. PDB ID: FPV039:Bik = 5TZP, Mcl-1:Bim = 2NL9.
Figure 7
Figure 7
CNP058 is a potent inhibitor of UV induced apoptosis. The ability of HeLa cells expressing green fluorescent protein (GFP) tagged virus encoded apoptosis inhibitors CNP058 or F1 or cytosolic GFP to undergo apoptosis was determined 6 h after UV irradiation via flow cytometry. The percentage of apoptotic cells in total GFP-positive cell population was determined by AV-PE/TO-PRO-3 staining. Data are representative of three independent experiments, error bars represent the standard error of the mean (SEM), n = 3. Unpaired Student’s two-tailed t-test was performed in Excel, * p < 0.05, NS: non-significant.
Figure 8
Figure 8
The role of the conserve NWGR motif. Detailed cartoon representation of the NWGR or equivalent motif in (A) CNP058:Bim BH3 domain complex, (B) FPV039:Bik BH3 domain complex and (C) Mcl-1:Bim BH3 domain complex. The view is into the conserved hydrophobic ligand binding groove formed by α-helices 2–5. Residues involved in interactions are shown as sticks and labelled. Hydrogen bonds and ionic interactions between pro-survival proteins and pro-apoptotic ligands are denoted as black dotted lines. The Asn residues in the NWGR motif in FPV039 and Mcl-1 as well as the Thr in the TWGR motif are labelled in red. PDB ID: FPV039:Bik = 5TZP, Mcl-1:Bim = 2NL9.
Figure 9
Figure 9
Cartoon representation of B-factor distribution of Cα atoms in (A) CNP058:Bim BH3 domain complex, (B) FPV039:Bik BH3 domain complex The view is into the conserved hydrophobic ligand binding groove formed by α-helices 2–5. BH3 domain ligands were removed for clarity. Colour are based on residues with low B-factor (blue) to high B-factor (red). PDB ID: FPV039:Bik = 5TZP.
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