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• W2606465385 abstract "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. 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. Programmed cell death or apoptosis is a highly organized and tightly regulated mechanism of cell suicide (1Kerr J.F. Wyllie A.H. Currie A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12826) Google Scholar, 2Youle R.J. Strasser A. The BCL-2 protein family: opposing activities that mediate cell death.Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3538) Google Scholar) and is evolutionary conserved in all multicellular organisms (3Vaux D.L. Haecker G. Strasser A. An evolutionary perspective on apoptosis.Cell. 1994; 76: 777-779Abstract Full Text PDF PubMed Scopus (690) Google Scholar). Apoptosis allows the regulation of cell and tissue homeostasis as well as the removal of impaired, unwanted, or diseased cells (4Green D.R. Kroemer G. The pathophysiology of mitochondrial cell death.Science. 2004; 305: 626-629Crossref PubMed Scopus (2811) Google Scholar). 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 (5Galluzzi L. Brenner C. Morselli E. Touat Z. Kroemer G. Viral control of mitochondrial apoptosis.PLoS Pathog. 2008; 4e1000018Crossref PubMed Scopus (337) Google Scholar). B-cell lymphoma 2 (Bcl-2) 3The abbreviations used are: Bcl-2B-cell lymphoma 2BHBcl-2 homologyBaxBcl-2-associated X, apoptosis regulatorBakBcl-2 antagonist/killerFPVfowlpox virusvBcl-2viral Bcl-2SAXSsmall-angle X-ray scatteringWAXSwide-angle X-ray scatteringITCisothermal titration calorimetryTFZtranslation function Z-scoreLLGlog-likelihood gainMLmaximum likelihoodHSH. sapiensGGG. gallus. proteins are the main regulators of apoptosis executed by the intrinsic pathway (2Youle R.J. Strasser A. The BCL-2 protein family: opposing activities that mediate cell death.Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3538) Google Scholar). The Bcl-2 protein family is divided into two subfamilies, the proapoptotic Bcl-2 and prosurvival Bcl-2 proteins (6Chipuk J.E. Moldoveanu T. Llambi F. Parsons M.J. Green D.R. The BCL-2 family reunion.Mol. Cell. 2010; 37: 299-310Abstract Full Text Full Text PDF PubMed Scopus (1170) Google Scholar), which are characterized by the presence of one or more of the four conserved Bcl-2 homology (BH) motifs (7Kvansakul M. Hinds M.G. Structural biology of the Bcl-2 family and its mimicry by viral proteins.Cell Death Dis. 2013; 4: e909Crossref PubMed Scopus (106) Google Scholar). 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 (8Westphal D. Kluck R.M. Dewson G. Building blocks of the apoptotic pore: how Bax and Bak are activated and oligomerize during apoptosis.Cell Death Differ. 2014; 21: 196-205Crossref PubMed Scopus (263) Google Scholar), leading to the release of apoptogenic mediators such as cytochrome c that ultimately activate caspases and lead to the destruction of the cell (4Green D.R. Kroemer G. The pathophysiology of mitochondrial cell death.Science. 2004; 305: 626-629Crossref PubMed Scopus (2811) Google Scholar). 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-xL, A1, Bcl-w, and Bcl-b (9Shamas-Din A. Brahmbhatt H. Leber B. Andrews D.W. BH3-only proteins: orchestrators of apoptosis.Biochim. Biophys. Acta. 2011; 1813: 508-520Crossref PubMed Scopus (261) Google Scholar). 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 (10Kvansakul M. Hinds M.G. The structural biology of BH3-only proteins.Methods Enzymol. 2014; 544: 49-74Crossref PubMed Scopus (41) Google Scholar). B-cell lymphoma 2 Bcl-2 homology Bcl-2-associated X, apoptosis regulator Bcl-2 antagonist/killer fowlpox virus viral Bcl-2 small-angle X-ray scattering wide-angle X-ray scattering isothermal titration calorimetry translation function Z-score log-likelihood gain maximum likelihood H. sapiens G. gallus. Premature host cell apoptosis plays a significant role in combating viral infections, and many viruses have evolved strategies to counter host cell apoptotic defenses (5Galluzzi L. Brenner C. Morselli E. Touat Z. Kroemer G. Viral control of mitochondrial apoptosis.PLoS Pathog. 2008; 4e1000018Crossref PubMed Scopus (337) Google Scholar). For instance, many viruses express prosurvival factors to extend the lifespan of infected host cells (11Danthi P. Viruses and the diversity of cell death.Annu. Rev. Virol. 2016; 3: 533-553Crossref PubMed Scopus (81) Google Scholar). Numerous large DNA viruses utilize sequence and/or structural mimics of prosurvival Bcl-2 to inhibit apoptosis (7Kvansakul M. Hinds M.G. Structural biology of the Bcl-2 family and its mimicry by viral proteins.Cell Death Dis. 2013; 4: e909Crossref PubMed Scopus (106) Google Scholar). Examples include γ-herpesviruses 68 M11 (12Wang G.H. Garvey T.L. Cohen J.I. The murine gammaherpesvirus-68 M11 protein inhibits Fas- and TNF-induced apoptosis.J. Gen. Virol. 1999; 80: 2737-2740Crossref PubMed Scopus (76) Google Scholar), adenovirus E1B19K (13Chiou S.K. Tseng C.C. Rao L. White E. Functional complementation of the adenovirus E1B 19-kilodalton protein with Bcl-2 in the inhibition of apoptosis in infected cells.J. Virol. 1994; 68: 6553-6566Crossref PubMed Google Scholar), and African swine fever virus A179L (14Brun A. Rivas C. Esteban M. Escribano J.M. Alonso C. African swine fever virus gene A179L, a viral homologue of bcl-2, protects cells from programmed cell death.Virology. 1996; 225: 227-230Crossref PubMed Scopus (94) Google Scholar). Among the poxviruses, a number of homologs of Bcl-2 have been identified including M11L from myxoma virus (15Graham K.A. Opgenorth A. Upton C. McFadden G. Myxoma virus M11L ORF encodes a protein for which cell surface localization is critical in manifestation of viral virulence.Virology. 1992; 191: 112-124Crossref PubMed Scopus (56) Google Scholar), F1L from vaccinia virus (16Wasilenko S.T. Stewart T.L. Meyers A.F. Barry M. Vaccinia virus encodes a previously uncharacterized mitochondrial-associated inhibitor of apoptosis.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 14345-14350Crossref PubMed Scopus (133) Google Scholar, 17Fischer S.F. Ludwig H. Holzapfel J. Kvansakul M. Chen L. Huang D.C. Sutter G. Knese M. Häcker G. Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and acts together with the early viral protein E3L to block virus-associated apoptosis.Cell Death Differ. 2006; 13: 109-118Crossref PubMed Scopus (60) Google Scholar) and variola virus (18Marshall B. Puthalakath H. Caria S. Chugh S. Doerflinger M. Colman P.M. Kvansakul M. Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim.Cell Death Dis. 2015; 6e1680Crossref PubMed Scopus (32) Google Scholar), sheeppox virus SPPV14 (19Okamoto T. Campbell S. Mehta N. Thibault J. Colman P.M. Barry M. Huang D.C. Kvansakul M. Sheeppox virus SPPV14 encodes a Bcl-2-like cell death inhibitor that counters a distinct set of mammalian proapoptotic proteins.J. Virol. 2012; 86: 11501-11511Crossref PubMed Scopus (36) Google Scholar), and deerpox virus DPV022 (20Banadyga L. Lam S.C. Okamoto T. Kvansakul M. Huang D.C. Barry M. Deerpox virus encodes an inhibitor of apoptosis that regulates Bak and Bax.J. Virol. 2011; 85: 1922-1934Crossref PubMed Scopus (37) Google Scholar, 21Burton D.R. Caria S. Marshall B. Barry M. Kvansakul M. Structural basis of deerpox virus-mediated inhibition of apoptosis.Acta Crystallogr. D Biol. Crystallogr. 2015; 71: 1593-1603Crossref PubMed Scopus (22) Google Scholar). Interestingly, structural studies showed that M11L, F1L, and DPV022 adopt a Bcl-2 fold despite a lack of detectable sequence similarity to Bcl-2 (21Burton D.R. Caria S. Marshall B. Barry M. Kvansakul M. Structural basis of deerpox virus-mediated inhibition of apoptosis.Acta Crystallogr. D Biol. Crystallogr. 2015; 71: 1593-1603Crossref PubMed Scopus (22) Google Scholar, 22Campbell S. Thibault J. Mehta N. Colman P.M. Barry M. Kvansakul M. Structural insight into BH3 domain binding of vaccinia virus antiapoptotic F1L.J. Virol. 2014; 88: 8667-8677Crossref PubMed Scopus (34) Google Scholar, 23Kvansakul M. Yang H. Fairlie W.D. Czabotar P.E. Fischer S.F. Perugini M.A. Huang D.C. Colman P.M. Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands.Cell Death Differ. 2008; 15: 1564-1571Crossref PubMed Scopus (179) Google Scholar, 24Kvansakul M. van Delft M.F. Lee E.F. Gulbis J.M. Fairlie W.D. Huang D.C. Colman P.M. A structural viral mimic of prosurvival Bcl-2: a pivotal role for sequestering proapoptotic Bax and Bak.Mol. Cell. 2007; 25: 933-942Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 25Douglas A.E. Corbett K.D. Berger J.M. McFadden G. Handel T.M. Structure of M11L: a myxoma virus structural homolog of the apoptosis inhibitor, Bcl-2.Protein Sci. 2007; 16: 695-703Crossref PubMed Scopus (71) Google Scholar). Functionally, M11L was shown to act by sequestering Bax and Bak (24Kvansakul M. van Delft M.F. Lee E.F. Gulbis J.M. Fairlie W.D. Huang D.C. Colman P.M. A structural viral mimic of prosurvival Bcl-2: a pivotal role for sequestering proapoptotic Bax and Bak.Mol. Cell. 2007; 25: 933-942Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), whereas F1L inhibits apoptosis by sequestering Bim (22Campbell S. Thibault J. Mehta N. Colman P.M. Barry M. Kvansakul M. Structural insight into BH3 domain binding of vaccinia virus antiapoptotic F1L.J. Virol. 2014; 88: 8667-8677Crossref PubMed Scopus (34) Google Scholar). Sequencing of the fowlpox virus genome and subsequent analysis identified the virus open reading frame 39 (FPV039) (26Afonso C.L. Tulman E.R. Lu Z. Zsak L. Kutish G.F. Rock D.L. The genome of fowlpox virus.J. Virol. 2000; 74: 3815-3831Crossref PubMed Scopus (278) Google Scholar) 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 (27Banadyga L. Gerig J. Stewart T. Barry M. Fowlpox virus encodes a Bcl-2 homologue that protects cells from apoptotic death through interaction with the proapoptotic protein Bak.J. Virol. 2007; 81: 11032-11045Crossref PubMed Scopus (50) Google Scholar). 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 (27Banadyga L. Gerig J. Stewart T. Barry M. Fowlpox virus encodes a Bcl-2 homologue that protects cells from apoptotic death through interaction with the proapoptotic protein Bak.J. Virol. 2007; 81: 11032-11045Crossref PubMed Scopus (50) Google Scholar, 28Banadyga L. Veugelers K. Campbell S. Barry M. The fowlpox virus BCL-2 homologue, FPV039, interacts with activated Bax and a discrete subset of BH3-only proteins to inhibit apoptosis.J. Virol. 2009; 83: 7085-7098Crossref PubMed Scopus (35) Google Scholar). 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. 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 μm in a final buffer comprising 20 mm trisodium citrate, pH 6.0, 200 mm 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 (Fig. 1). FPV039 engages several BH3-only proteins with high affinity including Bid (2 nm), Bim (15 nm), Hrk (24 nm), Bmf (16 nm), Puma (24 nm), Noxa (28 nm), and Bik (30 nm). In contrast, FPV039 only engaged Bad (653 nm) with moderate affinity. Furthermore, FPV039 bound Bak BH3 with 76 nm affinity and Bax BH3 with 76 nm affinity. Because several viral prosurvival proteins from the Poxviridae have been shown to form dimers (18Marshall B. Puthalakath H. Caria S. Chugh S. Doerflinger M. Colman P.M. Kvansakul M. Variola virus F1L is a Bcl-2-like protein that unlike its vaccinia virus counterpart inhibits apoptosis independent of Bim.Cell Death Dis. 2015; 6e1680Crossref PubMed Scopus (32) Google Scholar, 21Burton D.R. Caria S. Marshall B. Barry M. Kvansakul M. Structural basis of deerpox virus-mediated inhibition of apoptosis.Acta Crystallogr. D Biol. Crystallogr. 2015; 71: 1593-1603Crossref PubMed Scopus (22) Google Scholar, 23Kvansakul M. Yang H. Fairlie W.D. Czabotar P.E. Fischer S.F. Perugini M.A. Huang D.C. Colman P.M. Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands.Cell Death Differ. 2008; 15: 1564-1571Crossref PubMed Scopus (179) Google Scholar, 29Cooray S. Bahar M.W. Abrescia N.G. McVey C.E. Bartlett N.W. Chen R.A. Stuart D.I. Grimes J.M. Smith G.L. Functional and structural studies of the vaccinia virus virulence factor N1 reveal a Bcl-2-like anti-apoptotic protein.J. Gen. Virol. 2007; 88: 1656-1666Crossref PubMed Scopus (148) Google Scholar), we next investigated the oligomeric state of FPV039 using small angle X-ray scattering (SAXS) (Table 1). In the presence of BH3 domain peptides, FPV039 could be concentrated to higher concentration, allowing us to perform SAXS analysis of FPV039·Bak 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 (Fig. 2). The scattering conforms to a straight line in the low q region on the Guinier plot (Fig. 2A), 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 (Fig. 2). 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 FPV039·Bak BH3 domain complex (Fig. 2C).Table 1Small-angle X-ray scattering data collection and scattering-derived parametersFPV039·Bak BH3 domainData collection parameters InstrumentSAXS/WAXS beamline, Australian Synchrotron Beam geometry (μm)80 × 200 Wavelength (keV)12 q range (Å−1)0.011–0.619 Exposure time (s)1 (per frame; 17 frames) Concentration range (mg ml−1)0.31–7.35 Temperature (K)293.15Structural parametersaReported for 7.35 mg/ml. I(0) (from Guinier) (cm−1)0.10 ± 0.000 Rg (from Guinier) (Å)19.66 ± 0.06Molecular mass determinationaReported for 7.35 mg/ml. Partial specific volume (cm3 g−1)bDetermined with MULCh (74).0.737 Contrast (Δρ × 1010 cm−2)2.900 Mr (from I(0)) (Da)17,902 Calculated monomeric Mr from sequence (Da)20,022Software used Primary data reductionSAXS/WAXS beamline software Data processingPRIMUSa Reported for 7.35 mg/ml.b Determined with MULCh (74Whitten A.E. Cai S. Trewhella J. MULCh: modules for the analysis of small-angle neutron contrast variation data from biomolecular assemblies.J. Appl. Crystallogr. 2008; 41: 222-226Crossref Scopus (132) Google Scholar). Open table in a new tab 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 (Fig. 3A and Table 2) and Bik BH3 domains (Fig. 3B). 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 (Fig. 4, A and B). Interestingly, FPV039 features an extended loop between α5 and α6 that is only visible in the FPV039·Bik BH3 complex structure but not in the FPV039·Bmf 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 (30Kvansakul M. Wei A.H. Fletcher J.I. Willis S.N. Chen L. Roberts A.W. Huang D.C. Colman P.M. Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1.PLoS Pathog. 2010; 6e1001236Crossref PubMed Scopus (82) Google Scholar).Table 2Data collection and refinement statisticsFPV039·Bmf BH3FPV039·Bik BH3Data collection Space groupC2P43 No. of molecules in asymmetric unit2 + 21 + 1 Cell dimensions a, b, c (Å)123.60, 41.20, 67.8247.85, 47.85, 74.30 α, β, γ (°)90.00, 93.14, 90.0090.00, 90.00, 90.00 Wavelength (Å)0.95370.9537 Resolution (Å)46.91–1.65 (1.68–1.65)47.85–1.35 (1.37–1.35) No. unique reflections40,990 (1,971)36,750 (1,846) Rsym or Rmerge0.050 (1.087)0.070 (1.592) I/σI10.7 (1.1)25.79 (1.7) CC1/20.99 (0.68)1.00 (0.60) Wilson B-factor25.613.8 Completeness (%)99.9 (98.6)100 (100) Redundancy3.9 (3.9)14.5 (13.5)Refinement Resolution (Å)1.651.35 No. reflections40,94236,707 Rwork/Rfree0.1729/0.20670.1444/0.1716 No. atoms Protein2,6011,348 Ligand/ion613 Water298202 B-factors Protein39.1316.35 Ligand/ion57.9524.53 Water46.5329.94 r.m.s. deviations Bond lengths (Å)0.0070.008 Bond angles (°)0.770.87 Ramachandran statistics (%) Favored96.2698.08 Allowed3.741.92 Disallowed0.000.00 Open table in a new tab Figure 4FPV039 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 FPV039·Bik BH3 domain complex (C), FPV039·Bmf BH3 domain complex (D), A1·Bmf BH3 domain complex (E), and Mcl-1·Bax 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 (Figs. 4, C and D, and 5A). Inspection of the FPV039·Bmf BH3 complex interface (Fig. 4C) 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-85FPV039 and Asp-151Bmf, and the hydrogen bonds are formed by Tyr-141FPV039 and His-154Bmf, Asn-82FPV039 and Asp-151Bmf, Asp-79FPV039 and Gln-147Bmf, and Arg-65FPV039 and Thr-138Bmf. Similarly, the interface of FPV039 and the Bik BH3 domain (Fig. 4D) 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 FPV039·Bmf is conserved and formed by Arg-85FPV039–Asp-57Bik in the FPV039·Bik BH3 domain complex. A total of five hydrogen bonds are formed between Bik BH3 domain and FPV039, two of which are also observed in FPV039·Bmf including Tyr-141FPV039–Asn-61Bik and Asn-82FPV039–Asp-57Bik. In contrast, three of the hydrogen bonds are unique to FPV039·Bik and not found in the Bmf complex, and these are formed by Ser-50FPV039–Tyr-64Bik, Lys-80FPV039–Asp-57Bik, and Gln-75FPV039–Gln-48Bik. Numerous viruses have been shown to express proteins to specifically inhibit apoptosis (5Galluzzi L. Brenner C. Morselli E. Touat Z. Kroemer G. Viral control of mitochondrial apoptosis.PLoS Pathog. 2008; 4e1000018Crossref PubMed Scopus (337) Google Scholar, 7Kvansakul M. Hinds M.G. Structural biology of the Bcl-2 family and its mimicry by viral proteins.Cell Death Dis. 2013; 4: e909Crossref PubMed Scopus (106) Google Scholar), 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 (7Kvansakul M. Hinds M.G. Structural biology of the Bcl-2 family and its mimicry by viral proteins.Cell Death Dis. 2013; 4: e909Crossref PubMed Scopus (106) Google Scholar). Recognizable Bcl-2 mimics encoded by viruses include adenovirus E1B19K (13Chiou S.K. Tseng C.C. Rao L. White E. Functional complementation of the adenovirus E1B 19-kilodalton protein with Bcl-2 in the inhibition of apoptosis in infected cells.J. Virol. 1994; 68: 6553-6566Crossref PubMed Google Scholar), Kaposi sarcoma herpesvirus KsBcl-2 (31Cheng E.H. Nicholas J. Bellows D.S. Hayward G.S. Guo H.G. Reitz M.S. Hardwick J.M. 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