Structural Investigation of Orf Virus Bcl-2 Homolog ORFV125 Interactions with BH3-Motifs from BH3-Only Proteins Puma and Hrk

Numerous viruses have evolved sophisticated countermeasures to hijack the early programmed cell death of host cells in response to infection, including the use of proteins homologous in sequence or structure to Bcl-2. Orf virus, a member of the parapoxviridae, encodes for the Bcl-2 homolog ORFV125, a potent inhibitor of Bcl-2-mediated apoptosis in the host. ORFV125 acts by directly engaging host proapoptotic Bcl-2 proteins including Bak and Bax as well as the BH3-only proteins Hrk and Puma. Here, we determined the crystal structures of ORFV125 bound to the BH3 motif of proapoptotic proteins Puma and Hrk. The structures reveal that ORFV125 engages proapoptotic BH3 motif peptides using the canonical ligand binding groove. An Arg located in the structurally equivalent BH1 region of ORFV125 forms an ionic interaction with the conserved Asp in the BH3 motif in a manner that mimics the canonical ionic interaction seen in host Bcl-2:BH3 motif complexes. These findings provide a structural basis for Orf virus-mediated inhibition of host cell apoptosis and reveal the flexibility of virus encoded Bcl-2 proteins to mimic key interactions from endogenous host signalling pathways.


Introduction
Viruses utilize numerous immunomodulatory strategies to neutralize host cell apoptosis [1][2][3] to keep them viable for viral replication. Among the apoptosis regulators the B-cell lymphoma-2 (Bcl-2) family proteins are key to mitochondrial-mediated (or intrinsic) apoptosis [1,4]. The family of Bcl-2 proteins are recognized by the presence of up to four short sequence motifs, known as Bcl-2 homology (BH) motifs and are sub-divided into either pro-survival Bcl-2 or pro-apoptotic Bcl-2 proteins [5]. The pro-survival proteins in higher organisms include Bcl-2, Bcl-w, Bcl-x L , Mcl-1, A1 (Bfl-1) and Bcl-B [4] and are inactivated by the interactions with their pro-apoptotic Bcl-2 counterparts [5]. The proapoptotic Bcl-2 family are divided into two sub-groups: the multi-BH-motif pro-apoptotic Bcl-2 proteins, Bak, Bax and Bok; or the BH3-only proteins, which only feature the BH3 motif. The BH3-only group includes Bad, Bid, Bik, Bim, Bmf, Hrk, Noxa and Puma and apart from Bid are intrinsically disordered [5]. The BH3 region of the pro-apoptotic proteins interacts with the canonical ligand binding groove of pro-survival Bcl-2 proteins to neutralize their function [5,6]. This step is crucial to allowing pro-apoptotic Bak and Bax to launch apoptosis by permeabilizing the mitochondrial outer membrane (MOMP) [7,8] to release pro-apoptogenic factors such as cytochrome c from mitochondria that ultimately activate the caspase cascade to dismantle the cell [9].
ORF virus (ORFV) is a large double stranded DNA virus belonging to the genus para-poxviridae [19], a sub-genus of the larger cordopoxvirinae sub-family, which consists of nine other sub-geneses that infect vertebrates [20]. ORFV is an emerging zoonotic disease that primarily infects sheep and goats and causes contagious ecthyma [21] that can be transmitted to humans by direct contact [22]. ORFV infections are characterized by proliferative skin lesions in the lips, nostrils, oral mucosa and muzzle of animals [21]. ORFV encodes ORFV125, a potent inhibitor of mitochondrial-mediated intrinsic apoptosis [23][24][25] and does not harbor recognizable Bcl-2 homology motifs [23,25]. However, ORFV125 bears a hydrophobic C-terminal region necessary for translocating the protein to mitochondria. ORFV125 supresses UV-induced DNA fragmentation, cytochrome c release and subsequent activation of caspases [23]. Structural and biochemical studies of ORFV125 revealed that it adopts a domain-swapped dimeric topology in solution [25] similar to that observed in some other poxviral Bcl-2 proteins such as Vaccinia virus (VACV) and variola virus (VARV) F1L [26][27][28], deerpox virus DPV022 [29] and tanapoxvirus TANV16L [30], and only interacts with four BH3 motifs of cellular pro-apoptotic Bcl-2 proteins: Bax, Bak, Puma and Harakiri (Hrk) [25]. Interestingly, ORFV125 did not show any detectable affinity towards Bim BH3 motif peptide, the universal human Bcl-2 interactor [25]. Here, we report the structural investigation of ORFV125 bound to Puma and Hrk BH3 motif peptides and propose that the mechanism of action of ORFV125 involves inhibition of these four pro-apoptotic proteins.

Protein Expression and Purification
Codon optimized synthetic cDNA encoding for ORFV125 (Uniprot accession number W6EVU4) but with the 30 C-terminal residues deleted was cloned into the bacterial expression vector pGEX-6P-1 (GenScript, Piscataway, NJ, USA). Recombinant ORFV125 was expressed in C41(DE3) cells using 2YT medium supplemented with 1 mg/mL ampicillin at 37 • C whilst shaking to an OD 600 of 0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.75 mM to induce protein expression for 18 h at 20 • C. Cells were harvested by centrifugation at 5000 rpm (JLA 9.1000 rotor, Avanti J E, Beckman Coulter, Brea, CA, USA) for 20 min and re-suspended in 100 mL of lysis buffer A (50 mM Tris pH 8.0, 300 mM NaCl, 5 mM DTT (dithiothreitol) and 1% Tergitol. The cells were lysed using sonication (programme 7, Model 705 Sonic Dismembrator, Fisher Scientific, Waltham, MA, USA) and lysates were centrifuged in SS34 tubes at 18,000 rpm (JA-25.50 rotor, Beckman Coulter Avanti J-E) for 30 min. The supernatant was applied to a 5 mL glutathione sepharose 4B column (GE Healthcare, Chicago, IL, USA) equilibrated with buffer A and washed with 150 mL of buffer A. HRV 3C protease was added overnight at 4 • C to liberate the target protein, which was subsequently eluted using buffer A and concentrated using a centrifugal concentrator (3 kDa molecular weight cut-off, Amicon ® Ultra 15) to a final volume of 3 mL. ORFV125 was subjected to size exclusion chromatography on a Superdex S200 10/300 column mounted on an ÄKTA Pure system (GE Healthcare) equilibrated in 25 mM HEPES pH 7.5, 150 mM NaCl and 5 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride). The final sample purity was estimated to be greater than 95% based on SDS-PAGE analysis. Selected fractions were pooled and concentrated using a centrifugal concentrator with 3 kDa molecular weight cut-off (Amicon ® Ultra 15) to a final concentration of 6.4 mg/mL.

Crystallization and Structure Determination
Crystals for ORFV125: Puma BH3 and ORFV125: Hrk BH3 complexes were obtained by mixing ORFV125 with human Puma (Uniprot accession number Q96PG8, residues 130-155) or human Hrk 26-mer peptides (Uniprot accession number O00198, residues 26-51) that span the BH3 region of these proteins [31], using a 1:1.3 molar ratio as described [32] and concentrated using a centrifugal concentrator with a 3 kDa molecular weight cut-off (Amicon ® Ultra 0.5) to 6 mg/mL. Concentrated protein was immediately used for crystallization trials. Initial high throughput sparse matrix screening was performed using 96-well sitting drop trays (SWISSCI, Neuheim, Switzerland).
All diffraction data were collected at the Australian Synchrotron MX2 beamline [33] using an Eiger 16M detector with an oscillation range of 0.1 • per frame at a wavelength of 0.9537 Å. The diffraction data were integrated using DIALS [34] and scaled using AIMLESS [35]. A molecular replacement solution was obtained with PHASER [36] using the previously solved ORFV125: Bax structure [25] as the initial search model. The ORFV125: Hrk BH3 unit cell contained four molecules of ORFV125 and four Hrk BH3 peptides in the asymmetric unit, with a 40.3% solvent content and final translation function Zscore (TFZ) and log-likelihood gain (LLG) values of 46 and 6373, respectively. The final model of ORFV125: Hrk BH3 complex was built manually over several cycles using Coot [37]. Refinement was performed using PHENIX [38] with final R work /R free values of 0.225/0.274, 99% of residues in the favoured region of the Ramachandran plot and no outliers. ORFV125: Puma BH3 crystals were obtained in 0.2 M magnesium acetate, 25% PEG 3350. The crystals were flash cooled at −173 • C in mother liquor supplemented with 20% ethylene glycol as cryoprotectant. The ORFV125: Puma BH3 complex formed single rod-shaped crystals belonging to space group P2 1 2 1 2 1 with a = 48.87 Å, b = 69.44 Å, c = 91.28 Å, α = 90.00 • , β = 90.00 • , γ = 90.00 • in the monoclinic crystal system. Diffraction data collection, integration and scaling were performed as described above. Molecular replacement was performed using PHASER [36] as described above. ORFV125: Puma BH3 crystals contain two molecules of ORFV125 and two Puma BH3 peptides in the unit cell, with a 38.79% solvent content and final TFZ and LLG values of 25.6 and 721.0 respectively. The final model of ORFV125: Puma BH3 was built and refined as above with final R work /R free values of 24.5/28.5, and 99% of residues in Ramachandran favoured region of the plot and no outliers. All images for ORFV125: Hrk and ORFV125: Puma complexes were generated using PyMOL molecular graphic system version 1.8.6.0 (Schrödinger, LLC, New York, NY, USA). All raw images were deposited at the SBGridDB [39] using their PDB accession codes. All software were accessed through the SBGrid suite [40].

Results
We have previously shown that ORF virus-encoded Bcl-2 homolog ORFV125 has a restricted binding profile towards peptides spanning the BH3 motif (26-mer) of proapoptotic Bcl-2 proteins and binds only human Bak, Bax, Puma and Hrk BH3 motif peptides with nanomolar to sub micromolar affinities [25]. To investigate the molecular basis of the interaction with Puma and Hrk BH3 motifs with ORFV125 we crystalized ORFV25:Puma BH3 and ORFV125:Hrk BH3 complexes by reconstituting recombinantly expressed and purified ORFV125 with Puma and Hrk BH3 peptides. Crystals of ORFV125:Puma BH3 complex diffracted to 2.40 Å resolution, whereas ORFV125:Hrk BH3 complex diffracted to 2.00 Å ( Table 1, Supplementary File S1). Both complex structures were solved by molecular replacement using the previously determined structure of ORFV125:Bax BH3 (PDB ID 7ADT) [25] as a search model. For ORFV125:Puma BH3 complex a clear and continuous electron density map was observed for ORFV125 residues 5-61 and 65-133, and Puma BH3 motif residues 132-155 ( Figure 1A,C), with the remainder of the residues presumed to be disordered. ORFV125 adopts the conserved Bcl-2 fold with seven α-helices in a domain-swapped dimeric configuration (Figure 2A) where the α1 helix of one protomer is swapped with the neighbouring protomer to form the dimer interface as previously seen in other poxvirus genera such as VACV F1L [27,28] (Figure 2C), VARV F1L [26], DPV022 [29] and TANV16L [30]. A structural similarity analysis was performed using DALI [41], which identified DPV022 (PDB ID 4UF1) [29] as the closest viral Bcl-2 homolog of ORFV125 with an rmsd of 2.6 Å over 119 Cα atoms and human Mcl-1 (PDB ID 3PK1) [42] as the closest cellular Bcl-2 homolog with an rmsd of 2.7 Å over 97 Cα atoms. Puma BH3 is bound to ORFV125 via the canonical hydrophobic ligand binding groove that is formed by helices α2-α5 ( Figure 2A). The four canonical BH3 motif defining residues from Puma, I137, L141 and M144 and L148, engage four hydrophobic pockets of the canonical ORFV125 ligand binding groove ( Figure 3A).    Furthermore, Puma Y152 occupies a fifth hydrophobic pocket in ORFV125, which is formed by residues A39, V42, W133 and V137 ( Figure 4A). The hallmark of pro-survival Bcl-2 interactions with BH3 motifs is the presence of an ionic interaction between prosurvival Bcl-2 proteins and pro-apoptotic BH3 motif ligands between a conserved arginine in the BH1 motif and aspartate of the BH3-motif [4]. In ORFV125:Puma this interaction is formed between R87 ORFV125 and D146 Puma . An additional ionic interaction was observed between E80 ORFV125 and R142 Puma , and this interaction is supplemented by hydrogen bonds between E54 ORFV125 and Q140 Puma as well as between G86 ORFV125 and N149 Puma ( Figure 3A). The ORFV125:Hrk BH3 complex also showed clear and continuous electron density for ORFV125 residues 5-63 and 67-142 and Hrk BH3 motif residues 54-74 ( Figure 1B,D). Similar to the ORFV125:Puma complex, ORFV125:Hrk BH3 adopted a domain-swapped topology ( Figure 2B). The four conserved hydrophobic residues of the Hrk BH3 motif, T33, L37, L40 and L44, protrude into four hydrophobic pockets in the canonical, ORFV125 ligand binding groove. Furthermore, multiple ionic interactions were found between R87 ORFV125 and D42 Hrk , E80 ORFV125 and R26 Hrk , E54 ORFV125 and R36 Hrk , E46 ORFV125 and R47 Hrk . These salt bridges were supplemented by additional hydrogen bonds between R139 ORFV125 amine group and the main chain carbonyl group of M49 Hrk ( Figure 3B).

Discussion
Bcl-2 homologs are widely used amongst large DNA viruses to ensure viral proliferation and/or survival [2,13,43]. Amongst the poxviridae, the majority of genera have been shown to encode apoptosis inhibiting Bcl-2 homologs including the orthopoxviridae vaccinia virus F1L [27,28], variola F1L [26] and ectromelia viruses EMV025 [44], leporipoxviridae myxomavirus M11L [45], cervidpoxviridae deerpox virus DPV022 [29,46], capripoxviridae sheeppox virus SPPV14 [47,48], avipoxviridae fowlpoxvirus FPV039 [49] and canarypox virus CNP058 [50], yatapoxviridae tanapox virus TANV16L [30] and parapoxviridae orf virus ORFV125 [23][24][25]. Whilst many poxviridae encode for sequence, structural or functional prosurvival Bcl-2 homologs, considerable diversity exists amongst these proteins [2]. There are widely differing interaction profiles with host pro-apoptotic Bcl-2 and differences in overall structure and topology as well as detailed interactions at the atomic level, as might be expected from these highly sequence divergent Bcl-2-fold sequences [2,51]. Here we report the crystal structures of ORF virus-encoded Bcl-2 homolog ORFV125 in complex with BH3 motif peptides of host pro-apoptotic BH3-only proteins Puma and Hrk. A comparison of the interactions of Puma BH3 or Hrk BH3 with ORFV125 identified three conserved ORFV125 residues: E54, E80 and R87 in the binding groove which formed ionic interaction with the BH3 motif of pro-apoptotic Bcl-2 proteins. Among those interactions, the ionic interactions formed by ORF125 E80 and R87 are also conserved in the previously reported structure of ORFV125: Bax BH3 complex [25]. This suggests that ORFV125 residues E80 and R87 may be crucial mediators for BH3 motif peptide binding in the ORFV125 binding groove. Detailed analysis of the interaction between different poxvirus-encoded Bcl-2 proteins and host pro-apoptotic Bcl-2 reveals that whilst the BH1 motif is frequently not conserved in viral protein residues that occupy the structurally equivalent positions of the NWGR motif in the BH1 region of, for example, human Mcl-1 and Bcl-x L , it still performs important roles in the sequence divergent virus encoded Bcl-2 proteins. A comparison of ORFV125:Puma BH3 complex with TANV16L: Puma BH3 [30] reveals that the mode of interaction is nearly identical, despite the ORFV125:Puma interaction being approximately four-fold lower in affinity [25]. The hallmark interaction between the Arg residue from the NWGR motif and the Asp from the BH3 motif peptide is present in both complexes; the equivalent region of the BH1 motif in ORFV125 is formed by the sequence 'SPGR' whereas in TANV16L it is 'NDNR', with both contributing to binding and specificity [30] (Figure 3C). A similar observation was made for both sheeppox virus SPPV14 [48] and deerpox virus DPV022 [29], which utilize a different Arg to recapitulate the hallmark ionic interaction observed for mammalian pro-survival Bcl-2 proteins bound to BH3 motif bearing interactors [4]. These observations underscore the major importance of the BH1 equivalent region for determining interactions between Bcl-2 family proteins [4]. Mutational studies in the BH1 motif of human pro-survival Bcl-2 proteins abrogate BH3 binding [52]. Furthermore, ORFV125 and TANV16L complexes with Puma utilized the same four conserved hydrophobic residues of BH3 motif peptides to interact with four hydrophobic pockets of the binding groove. However, in ORFV:Puma BH3 a fifth hydrophobic pocket is used by residue Y152 PUMA , a feature that has been previously observed for Bax complexes of Mcl-1, Bcl-x L [42] and Bcl-2 [53], where Bax M74 is lodged in a comparable pocket. The engagement of a fifth hydrophobic pocket by Puma BH3 was unexpected, with comparable pocket engagement previously only seen for Bax BH3. A superimposition of the ORFV125 complexes of Bax BH3 and Puma BH3 shows that engagement is near identical ( Figure 4A,B,D), whilst closely resembling the binding of the Bax BH3 M74 to Mcl-1 ( Figure 4C). Beyond the use of a fifth hydrophobic pocket, Q140 PUMA forms a conserved hydrogen bonding interaction in both ORFV125 and TANV16L complexes. In contrast to Q140 PUMA , R142 PUMA formed an additional ionic interaction with E80 ORFV125 that is not seen in TANV16L:Puma BH3 complex ( Figure 3A).
Puma is important for p53-dependent and independent regulation of apoptosis against various stimuli, such as genotoxic stress, radiation induced apoptosis, toxins, deregulated oncogene expression and viral infection [54]. Unlike other BH3-only proteins, Puma coun-teracts five cellular pro-survival Bcl-2 proteins (Bcl-2, Bcl-x L , Bcl-w, Mcl-1 and A1), though not Bcl-B [55] as well as most vBcl-2 proteins, through high affinity interactions with its BH3 motif [13]. A combination of structural data and knowledge of the conserved interaction between Bcl-2 proteins and Puma BH3 has been widely used for design peptidomimetic drugs to mimic these interactions [56]. In contrast to Puma, the molecular interactions supporting binding of BH3 motif peptide Hrk with ORFV125 are somewhat different to its previously reported interaction with sheeppox virus Bcl-2 homolog SPPV14 [48] ( Figure 3D). The ORFV125:Hrk BH3 interaction is predominantly stabilized by four ionic interactions including the conserved ionic interaction formed by R87 ORF and D42 HRK ( Figure 2B). In contrast, this ionic interaction found in the SPPV14:Hrk complex is not canonical and represents an alternative mode by which to form an ionic interaction utilizing D42 [48]. Interestingly, despite featuring an additional two ionic interactions compared to the Puma complex, the Hrk interaction with ORFV125 is of similar affinity (1912 nM for Hrk vs. 1753 nM for Puma) [25]; however, in the absence of a detailed mutagenesis analysis we cannot quantify the relative contributions to the affinity of the additional ORFV125:Hrk ionic interactions.
Despite adopting a domain-swapped dimeric configuration, the overall fold of ORFV125 closely resembles the conserved Bcl-2 fold and provides a hydrophobic groove for biding BH3-motifs. Complexes of ORFV125:Puma BH3 or ORFV125:Hrk BH3 are nearly identical to those observed for other Bcl-2:BH3 peptide complexes. A comparison of the structural similarity between ORFV125 with cellular counterparts using DALI analysis [41] showed that the closest mammalian structural homolog of ORFV125 is Mcl-1 (PDB ID 3PK1) [42] with an rmsd value of 2.7 Å over 97 Cα atoms, whilst sharing 19% sequence identity. However, several interesting differences are observed in the protein-peptide interfaces of ORFV125:Puma BH3 or Hrk BH3 complexes as well as Mcl-1:Puma BH3 complex and sheeppox virus SPPV14:Hrk BH3 complex. The sequence-structure alignment of ORFV125 with Mcl-1 revealed that the regions harbouring the BH motifs feature significant sequence variation, mostly centred near the binding groove, and provide the structural basis for the selectivity variation observed for pro-apoptotic BH3 ligands, with the ORFV125 binding profile towards BH3 ligands being more restricted and only binding to Bak, Bax, Puma and Hrk with sub-micromolar affinity [25]. In contrast, Mcl-1 showed affinity for a broad range of BH3 ligands and binds to Bak, Bax, Puma and Hrk tightly with low nanomolar affinity [57]. These data imply that ORFV125 inhibits apoptosis mediated by these four pro-apoptotic proteins. However, the relative contributions to the overall inhibition of premature host cell death made by each of the interactors remains to be clarified. Bax and Bak are vital to mitochondrial rupture and release of pro-apoptotic factors from this organelle, while the BH3-only proteins Puma and Hrk have more specific roles in initiating apoptosis [57]. Inhibition of the action of Puma and Hrk would allow the major cellular pro-survival proteins to maintain cell viability during invasion by ORF virus, with ORFV125 likely to work in tandem with endogenous pro-survival Bcl-2 proteins to maintain host cell viability. Our structures provide detailed mechanistic insights to provide a platform for deciphering the molecular interactions underlying inhibition of premature host cell apoptosis during ORF virus infectivity and proliferation.  Data Availability Statement: Data supporting the findings of this manuscript are available from the corresponding authors upon reasonable request. Coordinate files were deposited at the Protein Data Bank (https://www.rcsb.org/) (accessed 1 July 2021) using accession codes 7P0U and 7P0S for ORFV125:Hrk BH3 and ORFV125:Puma BH3, respectively. The raw X-ray diffraction data were deposited at the SBGrid Data Bank [39] (https://data.sbgrid.org/data/) using their PDB accession codes 7P0U and 7P0S for ORFV125:Hrk BH3 and ORFV125:Puma BH3, respectively.