Crystal structures of ORFV125 provide insight into orf virus-mediated inhibition of apoptosis

Abstract

Premature apoptosis of cells is a strategy utilized by multicellular organisms to counter microbial threats. Orf virus (ORFV) is a large double-stranded DNA virus belonging to the poxviridae. ORFV encodes for an apoptosis inhibitory protein ORFV125 homologous to B-cell lymphoma 2 or Bcl-2 family proteins, which has been shown to inhibit host cell encoded pro-apoptotic Bcl-2 proteins. However, the structural basis of apoptosis inhibition by ORFV125 remains to be clarified. We show that ORFV125 is able to bind to a range of peptides spanning the BH3 motif of human pro-apoptotic Bcl-2 proteins including Bax, Bak, Puma and Hrk with modest to weak affinity. We then determined the crystal structures of ORFV125 alone as well as bound to the highest affinity ligand Bax BH3 motif. ORFV125 adopts a globular Bcl-2 fold comprising 7 α-helices, and utilizes the canonical Bcl-2 binding groove to engage pro-apoptotic host cell Bcl-2 proteins. In contrast with a previously predicted structure, ORFV125 adopts a domain-swapped dimeric topology, where the α1 helix from one protomer is swapped into a neighbouring unit. Furthermore, ORFV125 differs from the conserved architecture of the Bcl-2 binding groove and instead of α3 helix forming one of the binding groove walls, ORFV125 utilizes an extended α2 helix that comprises the equivalent region of helix α3. This results in a subtle variation of previously observed dimeric Bcl-2 architectures in other poxvirus and human encoded Bcl-2 proteins. Overall, our results provide a structural and mechanistic basis for orf virus-mediated inhibition of host cell apoptosis.

Keywords: Bcl-2; apoptosis; crystallography; orf virus; poxvirus; virology.

Figure 1.. Sequence alignment of ORFV125 with pro-survival Bcl-2 family members.

The sequences of orf virus apoptosis regulator ORFV125 (uniprot accession number: W6EVU4), vaccinia virus F1L (uniprot: O57173), myxomavirus M11L (uniprot: Q85295) and human Bcl-x_L (uniprot: Q07817) were aligned using MUSCLE [35]. Secondary structure elements are marked based on the crystal structure of ORFV125, and BH motifs of Bcl-x_L highlighted (BH4 (bright green), BH3 (red), BH1 (yellow), BH2 (turquoise), transmembrane region (TM)(grey)) and shown in bold [7]. The helical regions are marked ‘H’ and unstructured loops by a bar above the sequence, with highly conservative substitutions indicated by ‘:’ and conserved substitutions indicated by ‘.’.

Figure 2.. ORFV125 engages a range of BH3 motif peptides of pro-apoptotic Bcl-2 proteins.

Affinities of recombinant ORFV125 for BH3 motif peptides (26-mers, except for a Bid 34-mer and Bax 28-mers) were measured using ITC and the raw thermograms shown. K_D values (in nM) are the means of 3 experiments ± SD NB: no binding detected. The binding affinities are tabulated in Table 1.

Figure 3.. Cartoon representation of apo-ORFV125 and ORFV125 bound to Bax BH3 domain and comparison with other vBcl-2 dimeric structures.

(a) Structure of apo-ORFV125 (green). ORFV125 helices are labelled α1–α7. The view on the left in (a) is into the hydrophobic binding groove formed by helices α2–α5. On the right, the view is down the 2-fold symmetry axis between the domain-swapped α1 helices. (b) ORFV125 (green) in complex with the Bax BH3 peptide (magenta). (c) VACV F1L (cyan) in complex with the Bak BH3 domain (raspberry) [36]. (d) TANV16L (salmon) in complex with the Bax BH3 domain (magenta) [38] (e) DPV022 (gold) in complex with the Bax BH3 domain (magenta) [39]. The views in (b–d) are as in (a). In all cases, the view on the left is a single protomer and the right hand view the dimer.

Figure 4.. Detailed view of ORFV125 interactions with Bax BH3.

(a) Detailed view of the ORFV125: Bax BH3 interface. The ORFV125 surface, backbone and binding groove are shown in grey, green and yellow, respectively, while Bax BH3 is shown in magenta. The principle interacting residues of BaxBH3 are labelled in blue and those of ORFV125 in black. (b) 2Fo-Fc electron density map of ORFV125: Bax BH3 contoured at 1.5 σ. (c) Superimposition of the protein Cα backbone of apo-ORFV (green) and ORFV of the ORFV125: Bax BH3 complex with the Bax BH3 peptide removed (grey). The inset shows an enlarged view of the protein backbone movement of the ORFV125 ligand-binding groove upon binding to Bax BH3 peptide. (d) A cartoon representation of apo-BHRF1 (PDB ID 1Q59) is shown in yellow, BHRF1:Bim BH3 (PDB ID 2WH6) is shown in red. (e) A cartoon representation of apo-M11L (PDB ID 2JBX) is shown in chocolate, M11L:Bak BH3 (PDB ID 2JBY) is shown in sky blue.

Figure 5.. ORFV125 is a dimer in solution.

Size-exclusion chromatography of ORFV125 using a Superdex S75 3.2/300 column. The elution profile of the peak of interest (ORFV125) is 1.71 ml (black). The molecular mass standards shown are albumin (66 kDa) (blue), carbonic anhydrase (29 kDa) (grey) and cytochrome c (Cyt c) (12 kDa) (orange), AU: absorbance units at wavelength 280 nm.

Figure 6.. Comparison of the BH3 motif peptide binding profile of ORFV125 with other dimeric Bcl-2 homologues from other poxviruses.

Dimeric vBcl-2 proteins include vaccinia virus F1L (VACV F1L) (37), variola virus F1L (VARV F1L) [37], tanapox virus TANV16L [38], deerpox virus DPV022 [39]. (a) Binding profile of ORFV125 with BH3 motif peptides (b) binding profile of VACV F1L and (c) binding profile of VARV F1L, (d) binding profile of TANV16L and (e) binding profile of DPV022. The sequences of all BH3 motif peptides used in (a), (b) and (c) are of human origin. The line weight of the bars indicates the binding affinity ranges from 0 to 99 nM, <999 nM, and <1000 nM, as shown in the inset.