Crystal structures of the sheeppoxvirus encoded inhibitor of apoptosis SPPV14 bound to Hrk and Bax BH3 peptides

AbstractProgrammed death of infected cells is used by multicellular organisms to counter viral infections. Sheeppoxvirus encodes for SPPV14, a potent inhibitor of Bcl-2 mediated apoptosis. We reveal the structural basis of apoptosis inhibition by determining crystal structures of SPPV14 bound to BH3 motifs of proapoptotic Bax and Hrk. The structures show that SPPV14 engages BH3 peptides using the canonical ligand binding groove. Unexpectedly, Arg84 from SPPV14 forms an ionic interaction with the conserved Asp in the BH3 motif in a manner that replaces the canonical ionic interaction seen in almost all host Bcl-2:BH3 motif complexes. These results reveal the flexibility of virus encoded Bcl-2 proteins to mimic key interactions from endogenous host signalling pathways to retain BH3-binding and pro-survival functionality.

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Poxviral Strategies to Overcome Host Cell Apoptosis

Pathogens ◽

10.3390/pathogens10010006 ◽

2020 ◽

Vol 10 (1) ◽

pp. 6

Author(s):

Chathura D. Suraweera ◽

Mark G. Hinds ◽

Marc Kvansakul

Keyword(s):

Viral Infections ◽

B Cell Lymphoma ◽

Intrinsic Apoptosis ◽

Host Cell Apoptosis ◽

Successful Infection ◽

Infected Cells ◽

Host Virus Interactions ◽

Almost All ◽

Virus Interactions ◽

Multicellular Organisms

Apoptosis is a form of cellular suicide initiated either via extracellular (extrinsic apoptosis) or intracellular (intrinsic apoptosis) cues. This form of programmed cell death plays a crucial role in development and tissue homeostasis in multicellular organisms and its dysregulation is an underlying cause for many diseases. Intrinsic apoptosis is regulated by members of the evolutionarily conserved B-cell lymphoma-2 (Bcl-2) family, a family that consists of pro- and anti-apoptotic members. Bcl-2 genes have also been assimilated by numerous viruses including pox viruses, in particular the sub-family of chordopoxviridae, a group of viruses known to infect almost all vertebrates. The viral Bcl-2 proteins are virulence factors and aid the evasion of host immune defenses by mimicking the activity of their cellular counterparts. Viral Bcl-2 genes have proved essential for the survival of virus infected cells and structural studies have shown that though they often share very little sequence identity with their cellular counterparts, they have near-identical 3D structures. However, their mechanisms of action are varied. In this review, we examine the structural biology, molecular interactions, and detailed mechanism of action of poxvirus encoded apoptosis inhibitors and how they impact on host–virus interactions to ultimately enable successful infection and propagation of viral infections.

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Structural Insight into African Swine Fever Virus A179L-Mediated Inhibition of Apoptosis

Journal of Virology ◽

10.1128/jvi.02228-16 ◽

2017 ◽

Vol 91 (6) ◽

Cited By ~ 26

Author(s):

Suresh Banjara ◽

Sofia Caria ◽

Linda K. Dixon ◽

Mark G. Hinds ◽

Marc Kvansakul

Keyword(s):

Crystal Structures ◽

African Swine Fever Virus ◽

Three Dimensional ◽

African Swine Fever ◽

Fever Virus ◽

Structural Basis ◽

Dna Virus ◽

Content Type ◽

Antiapoptotic Proteins ◽

Apoptosis Inhibition

ABSTRACT Programmed cell death is a tightly controlled process critical for the removal of damaged or infected cells. Pro- and antiapoptotic proteins of the Bcl-2 family are pivotal mediators of this process. African swine fever virus (ASFV) is a large DNA virus, the only member of the Asfarviridae family, and harbors A179L, a putative Bcl-2 like protein. A179L has been shown to bind to several proapoptotic Bcl-2 proteins; however, the hierarchy of binding and the structural basis for apoptosis inhibition are currently not understood. We systematically evaluated the ability of A179L to bind proapoptotic Bcl-2 family members and show that A179L is the first antiapoptotic Bcl-2 protein to bind to all major death-inducing mammalian Bcl-2 proteins. We then defined the structural basis for apoptosis inhibition of A179L by determining the crystal structures of A179L bound to both Bid and Bax BH3 motifs. Our findings provide a mechanistic understanding for the potent antiapoptotic activity of A179L by identifying it as the first panprodeath Bcl-2 binder and serve as a platform for more-detailed investigations into the role of A179L during ASFV infection. IMPORTANCE Numerous viruses have acquired strategies to subvert apoptosis by encoding proteins capable of sequestering proapoptotic host proteins. African swine fever virus (ASFV), a large DNA virus and the only member of the Asfarviridae family, encodes the protein A179L, which functions to prevent apoptosis. We show that A179L is unusual among antiapoptotic Bcl-2 proteins in being able to physically bind to all core death-inducing mammalian Bcl-2 proteins. Currently, little is known regarding the molecular interactions between A179L and the proapoptotic Bcl-2 members. Using the crystal structures of A179L bound to two of the identified proapoptotic Bcl-2 proteins, Bid and Bax, we now provide a three-dimensional (3D) view of how A179L sequesters host proapoptotic proteins, which is crucial for subverting premature host cell apoptosis.

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

Viruses ◽

10.3390/v13071374 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1374

Author(s):

Chathura D. Suraweera ◽

Mark G. Hinds ◽

Marc Kvansakul

Keyword(s):

Cell Death ◽

Structural Investigation ◽

Potent Inhibitor ◽

Host Cells ◽

Orf Virus ◽

Structural Basis ◽

Signalling Pathways ◽

Ionic Interaction ◽

Host Cell Apoptosis ◽

Binding Groove

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.

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Structural basis ofDeerpox virus-mediated inhibition of apoptosis

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715009402 ◽

2015 ◽

Vol 71 (8) ◽

pp. 1593-1603 ◽

Cited By ~ 18

Author(s):

Denis R. Burton ◽

Sofia Caria ◽

Bevan Marshall ◽

Michele Barry ◽

Marc Kvansakul

Keyword(s):

Structural Analysis ◽

Ligand Binding ◽

Structural Basis ◽

Inhibitor Of Apoptosis ◽

Defence Mechanism ◽

Partial Obstruction ◽

Host Cell Apoptosis ◽

Infected Cells ◽

Binding Groove ◽

Innate Defence

Apoptosis is a key innate defence mechanism to eliminate virally infected cells. To counteract premature host-cell apoptosis, poxviruses have evolved numerous molecular strategies, including the use of Bcl-2 proteins, to ensure their own survival. Here, it is reported that theDeerpox virusinhibitor of apoptosis, DPV022, only engages a highly restricted set of death-inducing Bcl-2 proteins, including Bim, Bax and Bak, with modest affinities. Structural analysis reveals that DPV022 adopts a Bcl-2 fold with a dimeric domain-swapped topology and binds pro-death Bcl-2 proteinsviatwo conserved ligand-binding grooves found on opposite sides of the dimer. Structures of DPV022 bound to Bim, Bak and Bax BH3 domains reveal that a partial obstruction of the binding groove is likely to be responsible for the modest affinities of DPV022 for BH3 domains. These findings reveal that domain-swapped dimeric Bcl-2 folds are not unusual and may be found more widely in viruses. Furthermore, the modest affinities of DPV022 for pro-death Bcl-2 proteins suggest that two distinct classes of anti-apoptotic viral Bcl-2 proteins exist: those that are monomeric and tightly bind a range of death-inducing Bcl-2 proteins, and others such as DPV022 that are dimeric and only bind a very limited number of death-inducing Bcl-2 proteins with modest affinities.

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

Biochemical Journal ◽

10.1042/bcj20200776 ◽

2020 ◽

Vol 477 (23) ◽

pp. 4527-4541 ◽

Cited By ~ 1

Author(s):

Chathura D. Suraweera ◽

Mark G. Hinds ◽

Marc Kvansakul

Keyword(s):

Host Cell ◽

Crystal Structures ◽

B Cell Lymphoma ◽

Orf Virus ◽

Structural Basis ◽

Inhibitory Protein ◽

Affinity Ligand ◽

Host Cell Apoptosis ◽

Mechanistic Basis ◽

Binding Groove

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.

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Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

Biochemical Journal ◽

10.1042/bcj20190289 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3227-3240 ◽

Cited By ~ 1

Author(s):

Shanshan Wang ◽

Yanxiang Zhao ◽

Long Yi ◽

Minghe Shen ◽

Chao Wang ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Changes ◽

Magnaporthe Oryzae ◽

Structural Information ◽

Complex Structure ◽

Critical Role ◽

Catalytic Process ◽

Structural Basis ◽

Salt Bridges ◽

Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

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Immunosurveillance of Cancer and Viral Infections with Regard to Alterations of Human NK Cells Originating from Lifestyle and Aging

Biomedicines ◽

10.3390/biomedicines9050557 ◽

2021 ◽

Vol 9 (5) ◽

pp. 557

Author(s):

Xuewen Deng ◽

Hiroshi Terunuma ◽

Mie Nieda

Keyword(s):

Nk Cells ◽

Viral Infections ◽

Nk Cell ◽

Healthy Lifestyle ◽

Cell Activity ◽

Nk Cell Activity ◽

Effector Cells ◽

Cell Dysfunction ◽

Forest Bathing ◽

Infected Cells

Natural killer (NK) cells are cytotoxic immune cells with an innate capacity for eliminating cancer cells and virus- infected cells. NK cells are critical effector cells in the immunosurveillance of cancer and viral infections. Patients with low NK cell activity or NK cell deficiencies are predisposed to increased risks of cancer and severe viral infections. However, functional alterations of human NK cells are associated with lifestyles and aging. Personal lifestyles, such as cigarette smoking, alcohol consumption, stress, obesity, and aging are correlated with NK cell dysfunction, whereas adequate sleep, moderate exercise, forest bathing, and listening to music are associated with functional healthy NK cells. Therefore, adherence to a healthy lifestyle is essential and will be favorable for immunosurveillance of cancer and viral infections with healthy NK cells.

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Mass Spectrometry versus Conventional Techniques of Protein Detection: Zika Virus NS3 Protease Activity towards Cellular Proteins

Molecules ◽

10.3390/molecules26123732 ◽

2021 ◽

Vol 26 (12) ◽

pp. 3732

Author(s):

Agnieszka Dabrowska ◽

Aleksandra Milewska ◽

Joanna Ner-Kluza ◽

Piotr Suder ◽

Krzysztof Pyrc

Keyword(s):

Mass Spectrometry ◽

Zika Virus ◽

Viral Infections ◽

Protein Detection ◽

Extensive Discussion ◽

Protein Targets ◽

Highly Sensitive ◽

Infected Cells ◽

Ns3 Protease ◽

To Receive

Mass spectrometry (MS) used in proteomic approaches is able to detect hundreds of proteins in a single assay. Although undeniable high analytical power of MS, data acquired sometimes lead to confusing results, especially during a search of very selective, unique interactions in complex biological matrices. Here, we would like to show an example of such confusing data, providing an extensive discussion on the observed phenomenon. Our investigations focus on the interaction between the Zika virus NS3 protease, which is essential for virus replication. This enzyme is known for helping to remodel the microenvironment of the infected cells. Several reports show that this protease can process cellular substrates and thereby modify cellular pathways that are important for the virus. Herein, we explored some of the targets of NS3, clearly shown by proteomic techniques, as processed during infection. Unfortunately, we could not confirm the biological relevance of protein targets for viral infections detected by MS. Thus, although mass spectrometry is highly sensitive and useful in many instances, also being able to show directions where cell/virus interaction occurs, we believe that deep recognition of their biological role is essential to receive complete insight into the investigated process.

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Crystal Structures of the Human and Fungal Cytosolic Leucyl-tRNA Synthetase Editing Domains: A Structural Basis for the Rational Design of Antifungal Benzoxaboroles

Journal of Molecular Biology ◽

10.1016/j.jmb.2009.04.073 ◽

2009 ◽

Vol 390 (2) ◽

pp. 196-207 ◽

Cited By ~ 65

Author(s):

Elena Seiradake ◽

Weimin Mao ◽

Vincent Hernandez ◽

Stephen J. Baker ◽

Jacob J. Plattner ◽

...

Keyword(s):

Crystal Structures ◽

Rational Design ◽

Trna Synthetase ◽

Structural Basis

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Structural basis of adhesive binding by desmocollins and desmogleins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1606272113 ◽

2016 ◽

Vol 113 (26) ◽

pp. 7160-7165 ◽

Cited By ~ 74

Author(s):

Oliver J. Harrison ◽

Julia Brasch ◽

Gorka Lasso ◽

Phinikoula S. Katsamba ◽

Goran Ahlsen ◽

...

Keyword(s):

Amino Acids ◽

Crystal Structures ◽

Structural Basis ◽

Cellular Interactions ◽

Opposite Charge ◽

Charged Amino Acids ◽

Structural Framework ◽

Classical Cadherins ◽

Coated Bead

Desmosomes are intercellular adhesive junctions that impart strength to vertebrate tissues. Their dense, ordered intercellular attachments are formed by desmogleins (Dsgs) and desmocollins (Dscs), but the nature of trans-cellular interactions between these specialized cadherins is unclear. Here, using solution biophysics and coated-bead aggregation experiments, we demonstrate family-wise heterophilic specificity: All Dsgs form adhesive dimers with all Dscs, with affinities characteristic of each Dsg:Dsc pair. Crystal structures of ectodomains from Dsg2 and Dsg3 and from Dsc1 and Dsc2 show binding through a strand-swap mechanism similar to that of homophilic classical cadherins. However, conserved charged amino acids inhibit Dsg:Dsg and Dsc:Dsc interactions by same-charge repulsion and promote heterophilic Dsg:Dsc interactions through opposite-charge attraction. These findings show that Dsg:Dsc heterodimers represent the fundamental adhesive unit of desmosomes and provide a structural framework for understanding desmosome assembly.

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