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Programmed cell death or apoptosis of infected host cells is an important defense mechanism in response to viral infections. This process is regulated by proapoptotic and prosurvival members of the B-cell lymphoma 2 (Bcl-2) protein family. To counter premature death of a virus-infected cell, poxviruses use a range of different molecular strategies including the mimicry of prosurvival Bcl-2 proteins. One such viral prosurvival protein is the fowlpox virus protein FPV039, which is a potent apoptosis inhibitor, but the precise molecular mechanism by which FPV039 inhibits apoptosis is unknown.
To understand how fowlpox virus inhibits apoptosis, we examined FPV039 using isothermal titration calorimetry, small-angle X-ray scattering, and X-ray crystallography. Here, we report that the fowlpox virus prosurvival protein FPV039 promiscuously binds to cellular proapoptotic Bcl-2 and engages all major proapoptotic Bcl-2 proteins. Unlike other identified viral Bcl-2 proteins to date, FPV039 engaged with cellular proapoptotic Bcl-2 with affinities comparable with those of Bcl-2's endogenous cellular counterparts.
Structural studies revealed that FPV039 adopts the conserved Bcl-2 fold observed in cellular prosurvival Bcl-2 proteins and closely mimics the structure of the prosurvival Bcl-2 family protein Mcl-1. Our findings suggest that FPV039 is a pan-Bcl-2 protein inhibitor that can engage all host BH3-only proteins, as well as Bcl-2-associated X, apoptosis regulator (Bax) and Bcl-2 antagonist/killer (Bak) proteins to inhibit premature apoptosis of an infected host cell. This work therefore provides a mechanistic platform to better understand FPV039-mediated apoptosis inhibition. Introduction Programmed cell death or apoptosis is a highly organized and tightly regulated mechanism of cell suicide (, ) and is evolutionary conserved in all multicellular organisms. Apoptosis allows the regulation of cell and tissue homeostasis as well as the removal of impaired, unwanted, or diseased cells. In addition to organogenesis, homeostasis, and roles in development, apoptosis also acts as a host defense mechanism to remove infected cells during viral infection, thus preventing viral survival and proliferation.
B-cell lymphoma 2 (Bcl-2) proteins are the main regulators of apoptosis executed by the intrinsic pathway. The Bcl-2 protein family is divided into two subfamilies, the proapoptotic Bcl-2 and prosurvival Bcl-2 proteins , which are characterized by the presence of one or more of the four conserved Bcl-2 homology (BH) motifs. Proapoptotic Bcl-2 proteins are further subdivided into members that contain multiple BH domains including Bax and Bak and the BH3-only proteins, which only harbor a BH3 motif. Upon activation, proapoptotic Bak and Bax oligomerize to permeabilize the outer mitochondrial membrane , leading to the release of apoptogenic mediators such as cytochrome c that ultimately activate caspases and lead to the destruction of the cell. Bak and Bax are activated by the expression of proapoptotic BH3-only proteins including Bim, Bid, Noxa, and Puma, which are able to directly bind and activate Bak and Bax, or by neutralizing prosurvival Bcl-2 proteins such as Bcl-2, Mcl-1, Bcl-x L, A1, Bcl-w, and Bcl-b.
Structural studies have shown that the three subfamilies interact with each other in a BH3 motif-dependent manner where the BH3 motif of a proapoptotic protein will interact with an extended hydrophobic groove in a receiving prosurvival Bcl-2 protein. Premature host cell apoptosis plays a significant role in combating viral infections, and many viruses have evolved strategies to counter host cell apoptotic defenses. For instance, many viruses express prosurvival factors to extend the lifespan of infected host cells.
Numerous large DNA viruses utilize sequence and/or structural mimics of prosurvival Bcl-2 to inhibit apoptosis. Examples include γ-herpesviruses 68 M11 , adenovirus E1B19K , and African swine fever virus A179L. Among the poxviruses, a number of homologs of Bcl-2 have been identified including M11L from myxoma virus , F1L from vaccinia virus (, ) and variola virus , sheeppox virus SPPV14 , and deerpox virus DPV022 (, ). Interestingly, structural studies showed that M11L, F1L, and DPV022 adopt a Bcl-2 fold despite a lack of detectable sequence similarity to Bcl-2 (,). Functionally, M11L was shown to act by sequestering Bax and Bak , whereas F1L inhibits apoptosis by sequestering Bim.
Sequencing of the fowlpox virus genome and subsequent analysis identified the virus open reading frame 39 (FPV039) as a putative prosurvival Bcl-2 protein due to the presence of recognizable BH1 and BH2 motifs as well as a C-terminal transmembrane domain. Subsequent studies revealed that FPV039 is a potent inhibitor of apoptosis and localizes to the mitochondrial membrane via a C-terminal transmembrane domain. Mechanistically, FPV039 suppresses apoptosis triggered by overexpression of proapoptotic proteins including Bak and Bax and all the BH3-only proteins and was shown to immunoprecipitate with the BH3-only proteins Bim and Bik as well as Bak and activated Bax (, ). However, the precise molecular and structural basis of how FPV039 inhibits apoptosis remains to be determined. To address these questions, we investigated the binding of FPV039 to peptides encoding the BH3 domain from all proapoptotic Bcl-2 family members including Bak, Bax, and Bok and all the BH3-only proteins.
We then determined crystal structures of FPV039 bound to two of the identified interactors, Bmf and Bik BH3. Our studies showed that FPV039 is a highly promiscuous prosurvival viral Bcl-2 (vBcl-2) protein that binds to all BH3-only proteins as well as Bax and Bak and provide a mechanistic platform to understand FPV039-mediated apoptosis inhibition. Results To understand how FPV039 interacts with proapoptotic members of the Bcl-2 family of proteins, truncated FPV039 comprising the first 143 residues was recombinantly expressed in Escherichia coli and purified using a two-step purification method using affinity chromatography followed by size-exclusion chromatography. FPV039 exhibited low solubility, necessitating sample buffer optimization to achieve concentrations of 30 μ m in a final buffer comprising 20 m m trisodium citrate, pH 6.0, 200 m m sodium chloride. We then examined binding of FPV039 to peptides encompassing the BH3 domain of all proapoptotic Bcl-2 proteins using isothermal titration calorimetry (ITC). Unexpectedly, ITC revealed that FPV039 was able to bind to peptides of all BH3-only proteins as well as peptides from Bak and Bax but not Bok. FPV039 engages several BH3-only proteins with high affinity including Bid (2 n m), Bim (15 n m), Hrk (24 n m), Bmf (16 n m), Puma (24 n m), Noxa (28 n m), and Bik (30 n m).
In contrast, FPV039 only engaged Bad (653 n m) with moderate affinity. Furthermore, FPV039 bound Bak BH3 with 76 n m affinity and Bax BH3 with 76 n m affinity. FPV039 interacts with BH3 domains of all BH3-only proteins and Bak. Raw heats measured by ITC for FPV039 interactions with BH3 domain peptides of proapoptotic Bcl-2 proteins from H. Sapiens ( HS) and G. Gallus ( GG).
Because several viral prosurvival proteins from the Poxviridae have been shown to form dimers (, ), we next investigated the oligomeric state of FPV039 using small angle X-ray scattering (SAXS). In the presence of BH3 domain peptides, FPV039 could be concentrated to higher concentration, allowing us to perform SAXS analysis of FPV039Bak BH3 domain peptide complex at concentrations ranging from 0.31 to 7.35 mg/ml. The scattering curve profile is conserved throughout the concentration range tested, suggesting an absence of interparticle interference. The scattering conforms to a straight line in the low q region on the Guinier plot ( A), and the calculated radius of gyration does not vary significantly with the measured concentration range, suggesting an absence of significant concentration effects from the highest concentration. The molecular mass calculated from I(0) on the absolute scattering scale across the concentration range was ∼20.0 kDa, corresponding to an oligomerization state of ∼1 for the FPV039Bak BH3 domain complex ( C). FPV039 is a monomer in solution. A, log plot of FPV039(1–143) raw SAXS data.
Concentrations are in decreasing order starting at 7.35 mg/ml ( top) followed by 3.34, 1.56, 0.70, and 0.31 mg/ml. B, Guinier plots of FPV039 SAXS data. Concentrations are as in A. C, summary of the SAXS data and analysis of the oligomeric state of FPV039 in solution. To understand the structural basis of FPV039 interaction with the BH3 domain of proapoptotic Bcl-2 proteins, we determined the structures of FPV039 in complex with Bmf ( A and ) and Bik BH3 domains ( B). Although FPV039 has limited sequence identity to Bcl-2 proteins, FPV039 adopts a Bcl-2 fold comprising eight α-helices and harboring the conserved BH3 domain-binding groove observed in all the other prosurvival Bcl-2 members that is used to bind both Bmf and Bik BH3 domains (, A and B). Interestingly, FPV039 features an extended loop between α5 and α6 that is only visible in the FPV039Bik BH3 complex structure but not in the FPV039Bmf BH3 complex structure, suggesting considerable flexibility in the loop.
The closest structural homolog as identified by a Dali search is Mcl-1 (root mean square deviation of 0.92 Å over 107 Cα carbon atoms with 25% sequence identity), whereas BHRF1 is the closest vBcl-2 homolog. FPV039 interacts with BH3 domains from Bmf and Bik via a conserved hydrophobic groove. Shown are schematic representations of FPV039 ( cyan) in complex with Bik BH3 domain ( hot pink) ( A) or Bmf BH3 domain ( green) ( B). The view is into the conserved hydrophobic ligand-binding groove formed by α-helices 2–5. C–F, surface representations of the FPV039Bik BH3 domain complex ( C), FPV039Bmf BH3 domain complex ( D), A1Bmf BH3 domain complex ( E), and Mcl-1Bax BH3 domain complex ( F) interfaces. Prosurvival Bcl-2 protein surfaces are shown in gray.
Residues involved in interactions are shown as sticks and labeled. Interactions are denoted as black dotted lines. Protein Data Bank accession codes are as follows: A1, 2VOG; and Mcl-1, 3PK1. FPV039 engages the BH3 domain of proapoptotic Bcl-2 proteins using the canonical ligand-binding groove found in other prosurvival Bcl-2 proteins. The ligand-binding groove of FPV039 is formed by α2–α5 with the α5 helix forming the floor of the groove. FPV039 bound to the BH3 domains of Bmf and Bik by utilizing the four conserved hydrophobic residues in the BH3 domain as well as a conserved ionic interaction between Asp-151 (for Bmf) and Asp-57 (for Bik) from the BH3 domain and Arg-85 from FPV039 (, C and D, and A). Sequence alignments of FPV039 with Bcl-2 family members.
A, structure-based sequence alignment of FPV039 with related cellular Bcl-2 proteins Mcl-1 and A1 as well as the closest viral Bcl-2 protein BHRF1. The α-helical secondary structure elements indicated (denoted as H and labeled Helices 1–8) are based on FPV039. BH motifs 1–4 are marked underneath the aligned sequences. Hydrophobic residues involved in the interactions with BH3 domain peptides are highlighted in yellow, and residues that are involved in hydrogen bonds or ionic interactions are highlighted in cyan. Accession numbers are as follows: FPV039,; Mcl-1,; A1,; and BHRF1,. B, sequence alignment of BH3 domains used in affinity measurements. The four conserved hydrophobic residues in the BH3 motif are indicated by arrows and numbered.
The hydrophobic residues involved in hydrophobic interactions with FPV039 in the case of Bmf and Bik BH3 domains are highlighted in yellow. Residues that form polar contacts with FPV039 are highlighted in cyan for Bmf and Bik BH3 domains as is the conserved aspartic acid of the BH3 domains. Sequence accession numbers are shown next to each sequence. Inspection of the FPV039Bmf BH3 complex interface ( C) revealed that Bmf residues Ile-142, Leu-146, Ile-149, and Phe-153 protrude into four hydrophobic pockets on the FPV039-binding groove. Furthermore, four hydrogen bonds and a salt bridge are found at the interface with the salt bridge formed by Arg-85 FPV039 and Asp-151 Bmf, and the hydrogen bonds are formed by Tyr-141 FPV039 and His-154 Bmf, Asn-82 FPV039 and Asp-151 Bmf, Asp-79 FPV039 and Gln-147 Bmf, and Arg-65 FPV039 and Thr-138 Bmf.
Similarly, the interface of FPV039 and the Bik BH3 domain ( D) involves four hydrophobic residues of Bik (Phe-60, Ile-56, Leu-53, and Val-49), which protrude into four hydrophobic pockets on FPV039. Furthermore, the salt bridge observed in FPV039Bmf is conserved and formed by Arg-85 FPV039–Asp-57 Bik in the FPV039Bik BH3 domain complex. A total of five hydrogen bonds are formed between Bik BH3 domain and FPV039, two of which are also observed in FPV039Bmf including Tyr-141 FPV039–Asn-61 Bik and Asn-82 FPV039–Asp-57 Bik. In contrast, three of the hydrogen bonds are unique to FPV039Bik and not found in the Bmf complex, and these are formed by Ser-50 FPV039–Tyr-64 Bik, Lys-80 FPV039–Asp-57 Bik, and Gln-75 FPV039–Gln-48 Bik. Discussion Numerous viruses have been shown to express proteins to specifically inhibit apoptosis (, ), in particular the Bcl-2-mediated pathway, to ensure viral survival and proliferation. Some of these viral effector proteins harbor detectable sequence identity to members of the Bcl-2 family of proteins.
Recognizable Bcl-2 mimics encoded by viruses include adenovirus E1B19K , Kaposi sarcoma herpesvirus KsBcl-2 , Epstein-Barr virus BHRF1 , γ-herpesvirus 68 M11 , African swine fever virus A179L (, ), turkey herpesvirus vNR13 , and orf virus ORFV125 as well as the poxviral Bcl-2 proteins SPPV14 from sheeppox and CNP058 from canarypox virus. However, many other virulence factors, in particular from poxviruses, share very limited sequence identity to prosurvival Bcl-2 but retain the Bcl-2 fold. Examples of such virulence factors include deerpox virus DPV022 (, ), vaccinia virus (, ) and variola virus F1L , and myxoma virus M11L.
FPV039 was identified as a putative vBcl-2 in the fowlpox virus genome as it shares limited sequence identity with mammalian Bcl-2 proteins. Cellular studies showed that FPV039 is a potent apoptosis inhibitor where FPV039 was able to inhibit apoptosis induced by Bak and Bax as well as all the BH3-only proteins (, ). However, the molecular basis of apoptosis inhibition remained unclear. To address this, we examined the ability of FPV039 to bind to BH3 domains of all proapoptotic Bcl-2 members ( and B). FPV039 exhibited binding to peptides of all BH3-only proteins with low nanomolar affinities except for Bad, which was only bound with modest affinity (653 n m). Furthermore, FPV039 bound the BH3 domain of the proapoptotic executor protein Bak with 76 n m affinity and Bax BH3 with 76 n m affinity but did not exhibit binding to Bok BH3. These findings suggest that FPV039 is able to neutralize all key Bcl-2-mediated aspects of the intrinsic apoptosis pathway by preventing initiation of apoptosis by directly sequestering all BH3-only proteins while also engaging proapoptotic Bak and Bax.
Interestingly, Banadyga et al. previously determined that FPV039 is only able to immunoprecipitate the BH3-only proteins Bim and Bik as well as proapoptotic Bak and activated Bax. Although this report also revealed some evidence for additional interactions with Noxa, Bid, and Bmf, ultimately the authors concluded that there was insufficient evidence for an interaction. We have now shown biochemically that peptides spanning the BH3 domain from all BH3-only proteins are able to engage FPV039.
This discrepancy could be due to the use of peptides rather than full-length proteins, although we note that it has been previously shown that the isolated BH3 domain of BH3-only proteins recapitulates the bulk of the properties of their full-length counterparts. Considering the tight nanomolar affinity of FPV039 for some of the BH3-only proteins that were not observed to immunoprecipitate, this discrepancy is surprising. However, this issue is compounded by the observation that immunoprecipitation experiments for Bcl-2 protein interactions are influenced by the choice of detergent , and ultimately a different choice of detergent for the immunoprecipitation experiment may have yielded a different outcome. At the structural level, FPV039 displays certain features that are unusual in the wider Bcl-2 family.
Long loop regions are typically located in the loop connecting α1 and α2 such as in Bcl-2 and Bcl-x L, which both feature particularly long connecting loops. In contrast, FPV039 contains a long loop connecting α5 and α6, a feature only previously seen in Bcl-b and its mouse homolog Boo. However, despite the topological similarity to Bcl-b and Boo, FPV039 is highly promiscuous with respect to binding proapoptotic Bcl-2 proteins, whereas Bcl-b only binds Bim and Bax. For Boo, conflicting interaction studies have been published, suggesting that Boo is either unable to bind proapoptotic Bcl-2 family members or engages all of them with weak micro- or millimolar affinities. Among the vBcl-2 proteins, only BHRF1 displays a long connecting sequence, which is located between α1 and α2 and harbors a short additional helical sequence , with the remaining structures of vBcl-2 proteins displaying compact helical bundle folds with very short loops. In the cases of vaccinia and variola virus F1L (, ) and deerpox virus DPV022 , the loop connecting α1 and α2 is particularly short, resulting in a dimeric domain-swapped arrangement. Although a number of structures of prosurvival Bcl-2 proteins bound to BH3-only proteins have been determined, the structural bases of prosurvival Bcl-2 protein interactions with Bik and Bmf are not well characterized.
To date, no other structures of prosurvival Bcl-2 proteins with Bik have been solved, and the only complex structure involving Bmf that has been determined is the A1Bmf BH3 complex ( E). Interestingly, A1 binding to Bmf BH3 does not involve the hallmark ionic interaction between the conserved arginine from the NWGR sequence with the conserved aspartic acid from the BH3 domain that is observed in the FPV039Bmf complex. Furthermore, the numerous additional hydrogen bond interactions seen in FPV039Bmf complex are also absent in A1Bmf complex ( E) including interactions contributed by FPV039 Asp-79, Asn-82, and Arg-85, which are conserved between FPV039 and A1. Consistent with this, FPV039 engages Bmf with a higher affinity compared with A1 (17 and 160 n m, respectively). In contrast, Mcl-1 utilizes the same interactions to bind Bax as FPV039 ( F), consistent with the identification of Mcl-1 as the closest structural homolog of FPV039.
Our findings that FPV039 is able to bind to all BH3-only proteins as well as Bak and Bax has implications for the putative mechanism of apoptosis inhibition. The major cellular prosurvival Bcl-2 proteins Bcl-2, Bcl-x L, Bcl-w, Mcl-1, and A1 harbor distinct binding profiles for proapoptotic Bcl-2 proteins as shown by their ability to bind either the BH3-only protein Bad or Noxa. These overlapping but distinct binding profiles enable finely tuned and differential regulation of apoptosis in a cell. Interestingly, the binding profile for FPV039 for proapoptotic Bcl-2 proteins differs from its cellular counterparts in two significant aspects. FPV039 is able to engage both Bad and Noxa with measurable affinity, which is highly unusual among prosurvival Bcl-2 proteins. In particular, the high affinity interaction with Noxa BH3 domain is striking.
This feature is unique to FPV039. Although orf virus ORFV125 has been shown to engage Noxa by immunoprecipitation, the affinity for this interaction has never been determined. The only other Noxa-binding vBcl-2 protein is A179L, which displays only moderate affinity. Noxa has been shown to be a crucial contributor in defense mechanisms during viral infection where Noxa expression greatly increased in the cells exposed to double-stranded RNA, interferon (IFN), and virus (, ). The high-affinity interaction of FPV039 with Noxa that we found suggests that fowlpox virus is able to counter host defense mechanisms initiated via Noxa during viral infection. Furthermore, our data suggest that FPV039 is capable of preventing initiation of Bcl-2-mediated apoptosis by directly sequestering all BH3-only proteins while also engaging the proapoptotic executor proteins Bak and Bax to prevent perforation of the outer mitochondrial membrane. Consequently, FPV039 is in principle equipped to neutralize a vast range of apoptotic stimuli and constitutes a broad spectrum Bcl-2 inhibitor.
C Affinity measured in n m by fluorescence polarization (IC 50). A number of studies on virus-encoded Bcl-2 proteins revealed that the range of proapoptotic Bcl-2 proteins that they interact with varies considerably across the different proteins and that a number of molecular mechanisms of action are utilized to inhibit premature host cell apoptosis (, ).
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VBcl-2 members have been demonstrated to rely either on sequestration of Bax and Bak as shown for myxoma virus M11L , sequestration of Bim as shown for vaccinia virus F1L , or a mixed mode reliant on neutralization of both Bim and Bak as shown for Epstein-Barr virus BHRF1 (, ). In comparison with mammalian Bcl-2 proteins, vBcl-2 proteins typically interact with host proapoptotic proteins with considerably lower affinity and tend to target only a smaller subset of proapoptotic regulators. Certain vBcl-2 members such as vaccinia and variola virus F1L (, ) and deerpox virus DPV022 target only a very restricted subset of host proapoptotic proteins, whereas others are more promiscuous such as sheeppox virus SPPV14 and murine γ-herpesvirus 68 M11. Interestingly, FPV039 is only the second vBcl-2 proteins after A179L that has been shown to interact with all major host proapoptotic Bcl-2 proteins.
Furthermore, FPV039 engages host BH3-only proteins with high affinities, thus more closely resembling the behavior of cellular prosurvival Bcl-2 rather than other vBcl-2 members. We note that the affinity of FPV039 for Bak is lower compared with cellular Bcl-2 proteins; however, the relative importance of all the proapoptotic ligands identified for FPV039 remains to be clarified. Nonetheless, this remarkable promiscuity is in striking contrast to vaccinia virus F1L , which relies solely on the sequestration of Bim to prevent host cell apoptosis, underscoring the diversity of mechanisms utilized by viruses and their Bcl-2 homologs to subvert intrinsic apoptosis.
Interestingly, FPV039 is one of a small number of vBcl-2 proteins that is able to engage Bad BH3, albeit with modest affinity. This raises the prospect of targeting FPV039 with first-generation BH3 mimetics such as ABT-737 and ABT-263, which were designed as Bad BH3 mimetics and engage the same mammalian prosurvival Bcl-2 proteins as Bad. Although the utility of targeting vBcl-2 proteins for therapy remains to be established for the Poxviridae, the efficacy of such a strategy was recently demonstrated for Epstein-Barr virus BHRF1. A phylogenetic analysis of FPV039 and its homologs among the avipoxviruses reveals that they can be divided into two clades ( A) in agreement with previous analyses using the conserved core 4b protein and more recently DNA polymerase. Furthermore, the overall protein sequence conservation between the canarypox and fowlpox Bcl-2 homologs is lower at 39% compared with overall conservation across homologous proteins (53%) , suggesting that the vBcl-2 proteins are subject to higher evolutionary pressure. This view would be consistent with the role of FPV039 as an immune evasion protein that is subject to higher selective pressure as part of the host-pathogen interface. Interestingly, mapping of conserved residues within the hydrophobic binding groove of FPV039 on avipoxviral homologs such as CNP058 reveals that several key residues in the groove are not conserved in the Clade B virus canarypox virus and even in the proximal Clade A virus turkeypox virus ( B), suggesting that CNP058 is likely to harbor a different proapoptotic Bcl-2 ligand-binding profile compared with FPV039.
Expansion of the phylogenetic analysis to include chicken prosurvival proteins reveals that the closest chicken prosurvival Bcl-2 protein is Mcl-1 ( A). This finding is consistent with our identification of Mcl-1 as the closest structural homolog of FPV039 and the conservation of interactions between FPV039BH3 ligand and Mcl-1BH3 ligand complexes. Consequently, it is tempting to speculate that chicken Mcl-1 may have been the first prosurvival Bcl-2 protein to be acquired by an ancestral avipoxvirus. However, this hypothesis requires experimental verification. A, phylogenetic analysis of Bcl-2-like proteins in avipoxviruses. The tree comprises FPV039 encoded by fowlpox virus, CNP058 encoded by canarypox virus, and Bcl-2-like proteins from penguinpox virus, pigeonpox virus, and turkeypox virus.
Avipoxvirus clades were classed as A and B according to the nomenclature of Jarmin et al. Phylogeny analysis reveals that FPV039, pigeonpox Bcl-2-like, and penguinpox Bcl-2 locate to the same clade, whereas CNP058 and turkeypox Bcl-2 are found on a separate clade.
The scale bar represents the number of substitutions per site, and the branch annotation is the branch support values where only branch support values of more than 0.5 are shown. B, multiple sequence alignment of FPV039 with the other avipoxvirus Bcl-2 proteins. Conserved NWGR motifs are underlined. Hydrophobic residues of FPV039 that interact with BH3 domains of Bik and Bmf are highlighted in yellow.
Residues of FPV039 that form ionic interactions or hydrogen bonds with BH3 domains of Bik and Bmf are highlighted in cyan. Conserved residues in related Bcl-2-like proteins from avipoxviruses postulated to form hydrophobic interactions with cellular proapoptotic Bcl-2 proteins are highlighted in yellow, and residues postulated to form ionic interactions or hydrogen bonds are highlighted in cyan. Accession codes for sequences used are as follows: 1) FPV039,; 2) pigeonpox Bcl-2-like, A0A068EEI8; 3) penguinpox Bcl-2-like, A0A068EHE4; 4) turkeypox Bcl-2-like, A0A0M3ZJZ2; and 5) CNP058,. In summary, our findings reveal that FPV039 adopts a Bcl-2 fold similar to the mammalian prosurvival protein Mcl-1, which allows engagement of all BH3-only proteins as well as Bak and Bax with high affinity. Our results will allow a detailed dissection of the interactions between FPV039 and host proapoptotic regulators to establish whether or not all of them are required as well as to pinpoint which interactions are ultimately key for suppression of premature host cell apoptosis.
Measurement of interactions with BH3 peptides Affinities of FPV039 for different BH3 peptides were measured using a MicroCal iTC200 system (GE Healthcare) at 25 °C. The measurements were performed in 20 m m trisodium citrate, pH 6.0, 200 m m NaCl at a final protein concentration of 30 μ m.
BH3 domain peptides at concentration of 300 μ m were titrated into the protein sample using 19 injections of 2 μl/injection. All assays were performed in triplicates. Protein concentrations were measured using a UV spectrophotometer (Thermo Scientific) at a wavelength of 280 nm. BH3 peptides concentrations were calculated from the dry weight of peptide. Small angle X-ray scattering SAXS data were collected at the Australian Synchrotron SAXS/WAXS beamline using protocols described previously (, ). Briefly, FPV039 in complex with Bak BH3 at concentrations of 7.35, 3.34, 1.56, 0.70, and 0.31 mg/ml in 20 m m trisodium citrate, pH 6.0, 200 m m NaCl was measured in a q range between 0.025 and 0.6 Å −1 at 12 keV with a 1.6-m camera length at 20 °C and collected on a Pilatus 1M detector (Dectris). The extinction coefficient of the complex was 1.872.
Beam stop integration was used to achieve normalization. The data were absolute scaled using distilled water. The samples were measured in a 1.5-mm quartz capillary to minimize radiation damage and flowed past the beam while 30 × 1-s exposures were measured on samples and blanks. Software specific to the beamline was used to integrate, average, and calibrate the scattering images against water. The forward scattering ( I(0)) and radius of gyration ( R g) were determined by the Guinier approximation (, ).
Sample homogeneity and monodispersity were independently determined using SDS-PAGE analysis and size-exclusion chromatography. Details of the data collection are summarized in.
Phylogenetic analysis Protein sequences of avipoxvirus Bcl-2-like proteins (fowlpox, canarypox, turkeypox, penguinpox, and pigeonpox) and cellular Bcl-2 proteins including Mcl-1, Bcl-2, A1, Bcl-x L, and Bcl-w from human, mouse, chicken, turkey, and penguin were aligned using T-Coffee. The multiple sequence alignment was modified using Gblocks where Gblocks selects conserved blocks of sequences that lack poorly aligned positions and divergent regions. Due to highly divergent sequences in the analysis, a less stringent setting that allows smaller final blocks chosen by Gblocks was used.
The phylogenetic tree was generated using a maximum likelihood (ML) approach using the maximum likelihood (PhyML) program (, ). The ML analysis was bootstrapped with 100 replicates. The final representation of the tree was produced using the tree dynamics (TreeDyn) program. T-Coffee, Gblocks, PhyML, and TreeDyn were accessed via the phylogeny.fr.
Author contributions M. Contributed to acquisition of data, analysis and interpretation of data, and drafting and revising the article. Contributed to acquisition of data, analysis and interpretation of data, and drafting and revising the article. Contributed to analysis and interpretation of data and drafting and revising the article. Contributed to study conception and design, acquisition of data, analysis and interpretation of data, and drafting and revising the article.
This work was supported in part by National Health and Medical Research Council Australia Project Grant APP1007918 (to M. K.), Australian Research Council Fellowship FT130101349 (to M. K.), and a La Trobe University scholarship (to M. The authors declare that they have no conflicts of interest with the contents of this article. The atomic coordinates and structure factors (codes and ) have been deposited in the Protein Data Bank.
SAXS data were deposited at the SASBDB using accession code SASDC63.
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