Canonical Ligand Binding Groove Research Articles

Crystal Structures of Epstein-Barr Virus Bcl-2 Homolog BHRF1 Bound to Bid and Puma BH3 Motif Peptides.

Apoptosis is a powerful defense mechanism used by multicellular organisms to counteract viral infection. In response to premature host cell suicide, viruses have evolved numerous countermeasures to ensure cell viability to optimize their replication by encoding proteins homologous in structure and function to cellular pro-survival Bcl-2 proteins. Epstein–Barr virus (EBV), a member of the Gammaherpesviridae, encodes the Bcl-2 homolog BHRF1, a potent inhibitor of Bcl-2-mediated apoptosis. BHRF1 acts by directly targeting Bid and Puma, two proapoptotic proteins of the Bcl-2 family. Here, we determined the crystal structures of BHRF1 bound to peptides spanning the Bcl-2 binding motifs (Bcl-2 homology 3 motif, BH3) of Bid and Puma. BHRF1 engages BH3 peptides using the canonical ligand-binding groove of its Bcl-2 fold and maintains a salt bridge between an Arg residue with a conserved Asp residue in the BH3 motif mimicking the canonical ionic interaction seen in host Bcl-2:BH3 motif complexes. Furthermore, both Bid and Puma utilize a fifth binding pocket in the canonical ligand binding groove of BHRF1 to provide an additional hydrophobic interaction distinct from the interactions previously seen with Bak and Bim. These findings provide a structural basis for EBV-mediated suppression of host cell apoptosis and reveal the flexibility of virus encoded Bcl-2 proteins in mimicking key interactions from the endogenous host signaling pathways.

Structural Insight into KsBcl-2 Mediated Apoptosis Inhibition by Kaposi Sarcoma Associated Herpes Virus.

Numerous large DNA viruses have evolved sophisticated countermeasures to hijack the premature programmed cell death of host cells post-infection, including the expression of proteins homologous in sequence, structure, or function to cellular Bcl-2 proteins. Kaposi sarcoma herpes virus (KSHV), a member of the gammaherpesvirinae, has been shown to encode for KsBcl-2, a potent inhibitor of Bcl-2 mediated apoptosis. KsBcl-2 acts by directly engaging host pro-apoptotic Bcl-2 proteins including Bak, Bax and Bok, the BH3-only proteins; Bim, Bid, Bik, Hrk, Noxa and Puma. Here we determined the crystal structures of KsBcl-2 bound to the BH3 motif of pro-apoptotic proteins Bid and Puma. The structures reveal that KsBcl-2 engages pro-apoptotic BH3 motif peptides using the canonical ligand binding groove. Thus, the presence of the readily identifiable conserved BH1 motif sequence “NWGR” of KsBcl-2, as well as highly conserved Arg residue (R86) 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 KSHV 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.

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.

An insight into the transmission role of insect vectors based on the examination of gene characteristics of African swine fever virus originated from non-blood sucking flies in pig farm environments

BackgroundInsect vector transmitted pathogens from contaminated environments are a key potential risk for public health. Meanwhile, transmission by non-blood sucking flies needs to be considered. Sequencing and phylogenetic tree analyses were used to study African swine fever virus (ASFV) genes derived from flies collected from pig farms that were infected with ASFV. The major differential genes were analyzed the encoded proteins, particularly their conformation, physico-chemical features, and interactions identified by immunophenotyping.ResultsResults showed that the ASFV p72 and D117L genes from these non-blood sucking flies identified by morphology have high sequence similarity from ASFV genotype II strains, however, A179L is found in an independent cluster, with five amino acid substitutions; four of which are in a continuous sequence. Moreover, the binding of a BH3 peptide into a surface groove formed by α-helices of ASFV A179L from the non-blood sucking flies is consistent with that of representative ASFV genotype II strains, Georgia/2007.They only differ in the direction of spatial interaction of six conserved amino residues. Many hydrophilic amino residues are located at the canonical ligand-binding groove of A179L from flies, with hydrophobic amino residues located at the corresponding positions in A179L of the Georgia/2007.Furthermore, analysis of protein interactions by immunophenotyping revealed that both A179Ls have similar roles in regulating autophagy and apoptosis.ConclusionsIn conclusion, the main genes that differ between ASFV from flies and Georgia/2007 were similar in structure and protein interaction, while exhibiting differences in physico-chemical features and amino acid variations. Understanding the mechanical transmission characteristics of non-blood sucking flies is important.

Crystal structures of the sheeppox virus encoded inhibitor of apoptosis SPPV14 bound to the proapoptotic BH3 peptides Hrk and Bax.

Programmed death of infected cells is used by multicellular organisms to counter viral infections. Sheeppox virus 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 prosurvival functionality.

Fuzzy Interactions Between 14‐3‐3ζ And Tau Regulate Tau Aggregation

The microtubule associated protein tau is vital for elongation and stability of microtubules in the central nervous system. However, aberrant accumulation of tau amyloid fibrils in neurons is a character of a neurodegenerative pathology called Alzheimer’s disease. Extensive studies have revealed that several factors promote aggregation of tau, including mutations, polyanions, phosphorylation, and interactions with other proteins. Previous studies have shown that the conserved scaffolding protein family 14‐3‐3 proteins interact with tau in a phosphorylation dependent manner and 14‐3‐3 proteins promote tau aggregation. Crystal structure of 14‐3‐3/tau complex revealed that phosphorylated tau segments bind to the ligand binding grooves of 14‐3‐3 dimer. Consequently, it is promising that 14‐3‐3 specific binding ligands will reduce/inhibit tau aggregation. Indeed, rational designed inhibitors of 14‐3‐3/tau interaction have been found to reduce binding of PKA‐phosphorylated tau protein to 14‐3‐3ζ. Although thermodynamics characterization showed that non‐phosphorylated tau segments bind to 14‐3‐3 much weaker than phosphorylated tau segments, 14‐3‐3ζ also promotes non‐phosphorylated tau aggregation. In this work, we investigated the interactions between tau K18 construct and 14‐3‐3ζ. We found that non‐phosphorylated and PKA‐phosphorylated tau proteins showed similar aggregating propensity in the presence of 14‐3‐3ζ. Replacing Ser324 with Ala had little effect of tau aggregation mediated by 14‐3‐3ζ protein. We further saturated the 14‐3‐3ζ ligand binding grooves by a high affinity peptide R18 and found that the presence of R18 could not reduce tau aggregation. Molecular dynamics simulations showed that tau K18 interact with 14‐3‐3ζ via extensive fuzzy interactions. Our results suggest that besides binding to the canonical ligand binding groove, tau also interacts with 14‐3‐3ζ via nonspecific interactions which are responsible for tau aggregation and cannot be inhibited by 14‐3‐3ζ specific inhibitors.Support or Funding InformationThis work was supported by Hubei University of Technology

Crystal Structure of African Swine Fever Virus A179L with the Autophagy Regulator Beclin.

Subversion of programmed cell death-based host defence systems is a prominent feature of infections by large DNA viruses. African swine fever virus (ASFV) is a large DNA virus and sole member of the Asfarviridae family that harbours the B-cell lymphoma 2 or Bcl-2 homolog A179L. A179L has been shown to bind to a range of cell death-inducing host proteins, including pro-apoptotic Bcl-2 proteins as well as the autophagy regulator Beclin. Here we report the crystal structure of A179L bound to the Beclin BH3 motif. A179L engages Beclin using the same canonical ligand-binding groove that is utilized to bind to pro-apoptotic Bcl-2 proteins. The mode of binding of Beclin to A179L mirrors that of Beclin binding to human Bcl-2 and Bcl-xL as well as murine γ-herpesvirus 68. The introduction of bulky hydrophobic residues into the A179L ligand-binding groove via site-directed mutagenesis ablates binding of Beclin to A179L, leading to a loss of the ability of A179L to modulate autophagosome formation in Vero cells during starvation. Our findings provide a mechanistic understanding for the potent autophagy inhibitory activity of A179L and serve as a platform for more detailed investigations into the role of autophagy during ASFV infection.

A structural investigation of NRZ mediated apoptosis regulation in zebrafish

Bcl‐2 family proteins play a crucial role in regulating apoptosis, a process critical for development, eliminating damaged or infected cells, host‐pathogen interactions and in disease. Dysregulation of Bcl‐2 proteins elicits an expansive cell survival mechanism promoting cell migration, invasion and metastasis. Through a network of intrafamily protein– protein interactions Bcl‐2 family members regulate the release of cell death factors from mitochondria. NRZ is a novel zebrafish pro‐survival Bcl‐2 orthologue resident on mitochondria and the endoplasmic reticulum (ER). However, the mechanism of NRZ apoptosis inhibition has not yet been clarified. Here we examined the interactions of NRZ with pro‐apoptotic members of the Bcl‐2 family using a combination of isothermal calorimetry and mutational analysis of NRZ. We show that NRZ binds almost all zebrafish pro‐apoptotic proteins and displays a broad range of affinities. Furthermore, we define the structural basis for apoptosis inhibition of NRZ by solving the crystal structure of both apo ‐NRZ and a holo form bound to a peptide spanning the binding motif of the pro‐apoptotic zBad, a BH3‐only protein orthologous to mammalian Bad. The crystal structure of NRZ revealed that it adopts the conserved Bcl‐2 like fold observed for other cellular pro‐survival Bcl‐2 proteins and employs the canonical ligand binding groove to bind Bad BH3 peptide. NRZ engagement of Bad BH3 involves the canonical ionic interaction between NRZ R86 and Bad D104 and an additional ionic interaction between NRZ D79 and Bad R100, and substitution of either NRZ R86 or D79 to Ala reduces the binding to Bad BH3 tenfold or more. Our findings provide a detailed mechanistic understanding for NRZ mediated anti‐apoptotic activity in zebrafish by revealing binding to both Bad and Noxa, suggesting that NRZ is likely to occupy a unique mechanistic role in zebrafish apoptosis regulation by acting as a highly promiscuous pro‐apoptotic Bcl‐2 binder.Support or Funding InformationThis work was supported by the Australian Research Council (Fellowship FT130101349 to MK) and LaTrobe university (scholarship to CDS)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A structural investigation of NRZ mediated apoptosis regulation in zebrafish

Bcl-2 family proteins play a crucial role in regulating apoptosis, a process critical for development, eliminating damaged or infected cells, host-pathogen interactions and in disease. Dysregulation of Bcl-2 proteins elicits an expansive cell survival mechanism promoting cell migration, invasion and metastasis. Through a network of intra-family protein–protein interactions Bcl-2 family members regulate the release of cell death factors from mitochondria. NRZ is a novel zebrafish pro-survival Bcl-2 orthologue resident on mitochondria and the endoplasmic reticulum (ER). However, the mechanism of NRZ apoptosis inhibition has not yet been clarified. Here we examined the interactions of NRZ with pro-apoptotic members of the Bcl-2 family using a combination of isothermal calorimetry and mutational analysis of NRZ. We show that NRZ binds almost all zebrafish pro-apoptotic proteins and displays a broad range of affinities. Furthermore, we define the structural basis for apoptosis inhibition of NRZ by solving the crystal structure of both apo-NRZ and a holo form bound to a peptide spanning the binding motif of the pro-apoptotic zBad, a BH3-only protein orthologous to mammalian Bad. The crystal structure of NRZ revealed that it adopts the conserved Bcl-2 like fold observed for other cellular pro-survival Bcl-2 proteins and employs the canonical ligand binding groove to bind Bad BH3 peptide. NRZ engagement of Bad BH3 involves the canonical ionic interaction between NRZ R86 and Bad D104 and an additional ionic interaction between NRZ D79 and Bad R100, and substitution of either NRZ R86 or D79 to Ala reduces the binding to Bad BH3 tenfold or more. Our findings provide a detailed mechanistic understanding for NRZ mediated anti-apoptotic activity in zebrafish by revealing binding to both Bad and Noxa, suggesting that NRZ is likely to occupy a unique mechanistic role in zebrafish apoptosis regulation by acting as a highly promiscuous pro-apoptotic Bcl-2 binder.

Drosophila melanogaster Guk-holder interacts with the Scribbled PDZ1 domain and regulates epithelial development with Scribbled and Discs Large

Epithelial cell polarity is controlled by components of the Scribble polarity module, and its regulation is critical for tissue architecture and cell proliferation and migration. In Drosophila melanogaster, the adaptor protein Guk-holder (Gukh) binds to the Scribbled (Scrib) and Discs Large (Dlg) components of the Scribble polarity module and plays an important role in the formation of neuromuscular junctions. However, Gukh's role in epithelial tissue formation and the molecular basis for the Scrib-Gukh interaction remain to be defined. We now show using isothermal titration calorimetry that the Scrib PDZ1 domain is the major site for an interaction with Gukh. Furthermore, we defined the structural basis of this interaction by determining the crystal structure of the Scrib PDZ1-Gukh complex. The C-terminal PDZ-binding motif of Gukh is located in the canonical ligand-binding groove of Scrib PDZ1 and utilizes an unusually extensive network of hydrogen bonds and ionic interactions to enable binding to PDZ1 with high affinity. We next examined the role of Gukh along with those of Scrib and Dlg in Drosophila epithelial tissues and found that Gukh is expressed in larval-wing and eye-epithelial tissues and co-localizes with Scrib and Dlg at the apical cell cortex. Importantly, we show that Gukh functions with Scrib and Dlg in the development of Drosophila epithelial tissues, with depletion of Gukh enhancing the eye- and wing-tissue defects caused by Scrib or Dlg depletion. Overall, our findings reveal that Scrib's PDZ1 domain functions in the interaction with Gukh and that the Scrib-Gukh interaction has a key role in epithelial tissue development in Drosophila.

Structural and Functional Insight into Canarypox Virus CNP058 Mediated Regulation of Apoptosis

Programmed cell death or apoptosis is an important component of host defense systems against viral infection. The B-cell lymphoma 2 (Bcl-2) proteins family is the main arbiter of mitochondrially mediated apoptosis, and viruses have evolved sequence and structural mimics of Bcl-2 to subvert premature host cell apoptosis in response to viral infection. The sequencing of the canarypox virus genome identified a putative pro-survival Bcl-2 protein, CNP058. However, a role in apoptosis inhibition for CNP058 has not been identified to date. Here, we report that CNP058 is able to bind several host cell pro-death Bcl-2 proteins, including Bak and Bax, as well as several BH3 only-proteins including Bim, Bid, Bmf, Noxa, Puma, and Hrk with high to moderate affinities. We then defined the structural basis for CNP058 binding to pro-death Bcl-2 proteins by determining the crystal structure of CNP058 bound to Bim BH3. CNP058 adopts the conserved Bcl-2 like fold observed in cellular pro-survival Bcl-2 proteins, and utilizes the canonical ligand binding groove to bind Bim BH3. We then demonstrate that CNP058 is a potent inhibitor of ultraviolet (UV) induced apoptosis in a cell culture model. Our findings suggest that CNP058 is a potent inhibitor of apoptosis that is able to bind to BH3 domain peptides from a broad range of pro-death Bcl-2 proteins, and may play a key role in countering premature host apoptosis.