Figure 1.
Vaccinia virus (VACV) proteins A52 and B14 share a Bcl-2–like fold.
(A) A52 and (B) B14 are shown as ribbons, ramp coloured from blue (N terminus) to red (C terminus). The disulphide bond observed in B14 is shown as purple spheres. (C) The structure of the poxvirus Bcl-2–like protein N1 (cyan; PDB ID 2uxe). (D) Structure-based sequence alignment of the VACV Bcl-2–like proteins. Residues that are highly or moderately conserved (BLOSUM62 score) are coloured marine and light blue, respectively, and cysteine residues that form the disulphide bond observed in B14 are boxed and in purple face. Residues of A52 that encompass the ‘P13’ peptide and residues removed in the A52ΔC46 mutant, which does not bind TRAF6, are marked with a solid and a dashed line, respectively. The secondary structures of A52, B14 and N1 are shown above the sequences with α helices represented as cylinders.
Table 1.
Data collection statistics.
Table 2.
Refinement statistics.
Figure 2.
A52, B14 and N1 utilise a conserved face for dimerisation, but the 2-fold rotations that relate monomers of the dimers differ significantly.
(A) Superposition of A52 (orange), B14 (magenta) and N1 (cyan; PDB ID 2uxe). (B–D) The molecular surfaces of (B) A52, (C) B14 and (D) N1 are shown in white, residues that form intermolecular (dimer) contacts being coloured orange (A52), magenta (B14) or cyan (N1). The molecules are oriented as in (A). Cylinders represent the two-fold rotation axes of the respective dimers.
Table 3.
Structural and sequence similarity of poxvirus, herpesvirus and cellular Bcl-2–like proteins.
Figure 3.
Regions of A52 implicated in TRAF6 and IRAK2 interactions.
The A52 dimer is shown in ribbon representation with residues that encompass the ‘P13’ peptide (residues 125–135) coloured orange and residues removed in the A52ΔC46 mutant (residues 145–189) coloured blue. While the putative IRAK2-interacting loop is exposed on the surface of A52, residues 145–189 form part of the core of the protein. (Left inset) The molecular surface of A52ΔC46 is shown in white, with A52 residues removed in this mutant (residues 145–189) shown as a blue ribbon. (Right inset) The structures of A52, B14 and N1 are shown superposed. Thicker coloured tubes denote the regions equivalent to residues 125–135, encompassing the ‘P13’ peptide, of A52 (orange): B14 residues 89–102 (magenta) and N1 residues 63–70 (cyan).
Figure 4.
The relative effects of A52, B14, N1 and M11 on signalling to NF-κB.
(A) VACV Bcl-2–like proteins affect signalling to NF-κB downstream of IL-1α and TNFα differently. HEK 293 cells were transfected with FLAG-tagged expression alleles for A52, B14, N1, M11 or empty vector (pCI; EV) together with an NF-κB reporter plasmid and Renilla internal control. Cells were treated with 100 ng/ml of IL-1α, TNFα or left untreated for 8 h, lysed and assayed for NF-κB–inducible luciferase activity. (B) B14, but not A52, blocks activation of NF-κB downstream of TRAF2 and TRAF6. HEK 293 cells were transfected with FLAG-tagged expression alleles for A52, B14, N1, M11 or empty vector (pCI; EV) together with plasmids for NF-κB reporter gene, Renilla internal control and either TRAF2 or TRAF6. Cells were lysed 24 h post-transfection and measured for NF-κB–inducible luciferase activity. (C) The N terminus of A52 is not required for inhibition of NF-κB downstream of IL-1α. HEK 293 cells were transfected with pOPINE vectors containing C-terminally His-tagged full length A52 or C-terminally His-tagged A52 lacking residues 1–36 (A52ΔN36), FLAG-tagged B14 or empty vector (pOPINE; EV) together with NF-κB reporter and Renilla internal control. Cells were treated as in (A). Data are expressed as means±standard deviation of 2–4 independent experiments. Statistics: two-tailed Student's t-Test (*P<0.05, **P<0.005 ***P<0.0005).
Figure 5.
A52 and B14 lack a surface BH3-peptide binding groove and do not block apoptosis.
(A) HeLa cells were transfected with vectors expressing FLAG-tagged A52, B14, N1, M11, untagged Bcl-xL or empty control vector (pCI; EV) together with CD20 expression vector. After a further 24 h, cells were treated with 0.5 µM staurosporine for 1 h or left untreated and assayed for mitochondrial dysfunction by JC-1 staining and FACS analysis. Data are expressed as means±standard deviation of 3 independent experiments. Statistics: two-tailed Student's t-Test (*P<0.05, **P<0.005). (B and C) The BH3-peptide binding groove of M11 is occluded in both A52 and B14. The structures of (B) A52 (orange ribbon) and (C) B14 (magenta ribbon) are shown superposed upon the structure of myxoma virus M11 (light grey ribbon) in complex with the BH3 peptide of human Bak-2 (lime green helix; PDB ID 2jby). Side chains that block the BH3-peptide binding groove are shown as sticks. (D) Stereogram depicting superposed Cα traces of A52 (orange), B14 (magenta), VACV N1 (cyan; PDB ID 2uxe), myxoma virus M11 (light grey; PDB IDs 2jbx and 2jby), mouse Bcl-xL (yellow; PDB IDs 1pq0 and 1pq1) and Epstein-Barr virus BHRF1 (dark blue; PDB ID 1q59). Two conformations of M11 and Bcl-xL are shown (in the presence and absence of bound BH3 peptide). The BH3 peptide of human Bak-2 in complex with myxoma virus M11 is shown (lime green helix; PDB ID 2jby).
Figure 6.
Structure-based phylogenetic analysis of virus and cellular Bcl-2–like proteins.
The structures were superposed and a pairwise distance matrix was constructed as described in Materials and Methods. PDB codes for each structure used are given followed by their species of origin in parentheses [Human (H), Mouse (M), Rat (R), C. elegans (C), Vaccinia virus (VACV), Myxoma virus (MV), Kaposi sarcoma herpes virus (KSHV), murine gamma-herpesvirus 68 (MHV), Epstein-Barr virus (EBV)]. Structures determined by X-ray crystallography are labelled in black, NMR models in grey and structures of Bcl-2–like proteins in complex with BH3 peptides are italicised. It is not certain that all Bcl-2–like proteins share a common ancestor and, as such, the root of the tree is shaded. Ribbon diagrams of representative structures for each protein are colour ramped from blue (N terminus) to red (C terminus).