Insight into the binding properties of MEKK3 PB1 to MEK5 PB1 from its solution structure

MEKK3 is a mitogen-activated protein kinase kinase kinase that participates in various signaling pathways. One of its functions is to activate the ERK5 signal pathway by phosphorylating and activating MEK5. MEKK3 and MEK5 each harbors a PB1 domain in the N-terminus, and they form a heterodimer via PB1-PB1 domain interaction that was reported to be indispensable to the activation of MEK5. Using NMR spectroscopy, we show here that a prolyl isomerization of the Gln38-Pro39 bond is present in MEKK3 PB1, which is the first case of structural heterogeneity within PB1 domains. We have solved the solution structures of both isomers and found a major difference between them in the Pro39 region. Residues Gly37-Leu40 form a type VIb beta-turn in the cis conformation, whereas no obvious character of beta-turn was observed in the trans conformation. Backbone dynamics studies have unraveled internal motions in the beta3/beta4-turn on a microsecond-millisecond time scale. Further investigation of its binding properties with MEK5 PB1 has demonstrated that MEKK3 PB1 binds MEK5 PB1 tightly with a Kd of about 10(-8) M. Mutagenesis analysis revealed that residues in the basic cluster of MEKK3 PB1 contributes differently to the PB1-PB1 interaction. Residues Lys 7 and Arg 5 play important roles in the interaction with MEK5 PB1. Taken together, this study provides new insights into structural details of MEKK3 PB1 and its binding properties with MEK5 PB1.     

Crystal structure of the PB1 domain of NBR1

The scaffold protein NBR1 is involved in signal transmission downstream of the serine/protein kinase from the giant muscle protein titin. Its N-terminal Phox and Bem1p (PB1) domain plays a critical role in mediating protein-protein interactions with both titin kinase and with another scaffold protein, p62. We have determined the crystal structure of the PB1 domain of NBR1 at 1.55A resolution. It reveals a type-A PB1 domain with two negatively charged residue clusters. We provide a structural perspective on the involvement of NBR1 in the titin kinase signalling pathway.     

Molecular recognition in dimerization between PB1 domains

The PB1 (Phox and Bem 1) domain is a recently identified module that mediates formation of a heterodimeric complex with other PB1 domain, e.g. the complexes between the phagocyte oxidase activators p67phox and p40phox and between the yeast polarity proteins Bem1p and Cdc24p. These PB1 domains harbor either a conserved lysine residue on one side or an acidic OPCA (OPR/PC/AID) motif around the other side; the lysine of p67phox or Bem1p likely binds to the OPCA of p40phox or Cdc24p, respectively, via electrostatic interactions. To further understand molecular recognition by PB1 domains, here we investigate the interactions mediated by proteins presenting both the lysine and OPCA on a single PB1 domain, namely Par6, atypical protein kinase C (aPKC), and ZIP. Par6 and aPKC form a complex via the interaction of the Par6 lysine with aPKC-OPCA but not via that between the aPKC lysine and Par6-OPCA, thereby localizing to the tight junction of epithelial cells. aPKC also uses its OPCA to interact with ZIP, another protein that has a PB1 domain presenting both the lysine and OPCA, whereas aPKC binds via the conserved lysine to MEK5 in the same manner as ZIP interacts with MEK5. In addition, ZIP can form a homotypic complex via the conserved electrostatic interactions. Thus the PB1 domain appears to be a protein module that fully exploits its two mutually interacting elements in molecular recognition to expand its repertoire of protein-protein interactions.     

The proapoptotic influenza A virus protein PB1-F2 regulates viral polymerase activity by interaction with the PB1 protein

The 11^th influenza A virus protein PB1-F2 was previously shown to enhance apoptosis in response to cytotoxic stimuli. The 87 amino acid protein that is encoded by an alternative reading frame of the PB1 polymerase gene was described to localize to mitochondria consistent with its proapoptotic function. However, PB1-F2 is also found diffusely distributed in the cytoplasm and in the nucleus suggesting additional functions of the protein. Here we show that PB1-F2 colocalizes and directly interacts with the viral PB1 polymerase protein. Lack of PB1-F2 during infection resulted in an altered localization of PB1 and decreased viral polymerase activity. Consequently, mutant viruses devoid of a functional PB1-F2 reading frame exhibited a small plaque phenotype. Thus, we have identified a novel function of PB1-F2 as an indirect regulator of the influenza virus polymerase activity via its interaction with PB1.     

Insight into molecular interactions between two PB1 domains

Specific protein-protein interactions play crucial roles in the regulation of any biological process. Recently, a new protein-protein interaction domain termed PB1 (Phox and Bem1) was identified, which is conserved throughout evolution and present in diverse proteins functioning in signal transduction, cell polarity and survival. Here, we investigated the structure and molecular interactions of the PB1 heterodimer complex composed of the PB1 domains of the yeast proteins Bem1 and Cdc24. A structural model of the Cdc24 PB1 was built by homology modeling and molecular dynamics simulations, and experimentally validated by 15N nuclear Overhauser effect spectroscopy (NOESY)-heteronuclear single quantum coherence (HSQC) analysis. Residues at the interface of the complex for both proteins were identified by NMR titration experiments. A model of the heterodimer was obtained by docking of the two PB1 domains with HADDOCK, which applies ambiguous interaction restraints on residues at the interface to drive the docking procedure. The refined model was validated by site-directed mutagenesis on both Bem1 and Cdc24. Finally, the docking was repeated from the newly published NMR structure of Cdc24, allowing us to assess the performance of homology-based docking. Our results provide insight into the molecular structure of the Bem1-Cdc24 PB1-mediated heterodimer, which allowed identification of critical residues at the binding interface.     

cis-Acting packaging signals in the influenza virus PB1, PB2, and PA genomic RNA segments

The influenza A virus genome consists of eight negative-sense RNA segments. The cis-acting signals that allow these viral RNA segments (vRNAs) to be packaged into influenza virus particles have not been fully elucidated, although the 5' and 3' untranslated regions (UTRs) of each vRNA are known to be required. Efficient packaging of the NA, HA, and NS segments also requires coding sequences immediately adjacent to the UTRs, but it is not yet known whether the same is true of other vRNAs. By assaying packaging of genetically tagged vRNA reporters during plasmid-directed influenza virus assembly in cells, we have now mapped cis-acting sequences that are sufficient for packaging of the PA, PB1, and PB2 segments. We find that each involves portions of the distal coding regions. Efficient packaging of the PA or PB1 vRNAs requires at least 40 bases of 5' and 66 bases of 3' coding sequences, whereas packaging of the PB2 segment requires at least 80 bases of 5' coding region but is independent of coding sequences at the 3' end. Interestingly, artificial reporter vRNAs carrying mismatched ends (i.e., whose 5' and 3' ends are derived from different vRNA segments) were poorly packaged, implying that the two ends of any given vRNA may collaborate in forming specific structures to be recognized by the viral packaging machinery.     

Mutational analyses of packaging signals in influenza virus PA, PB1, and PB2 genomic RNA segments

The influenza A virus genome consists of eight negative-sense RNA segments that must each be packaged to produce an infectious virion. We have previously mapped the minimal cis-acting regions necessary for efficient packaging of the PA, PB1, and PB2 segments, which encode the three protein subunits of the viral RNA polymerase. The packaging signals in each of these RNAs lie within two separate regions at the 3′ and 5′ termini, each encompassing the untranslated region and extending up to 80 bases into the adjacent coding sequence. In this study, we introduced scanning mutations across the coding regions in each of these RNA segments in order to finely define the packaging signals. We found that mutations producing the most severe defects were confined to a few discrete 5′ sites in the PA or PB1 coding regions but extended across the entire (80-base) 5′ coding region of PB2. In sequence comparisons among more than 580 influenza A strains from diverse hosts, these highly deleterious mutations were each found to affect one or more conserved bases, though they did not all lie within the most broadly conserved portions of the regions that we interrogated. We have introduced silent and conserved mutations to the critical packaging sites, which did not affect protein function but impaired viral replication at levels roughly similar to those of their defects in RNA packaging. Interestingly, certain mutations showed strong tendencies to revert to wild-type sequences, which implies that these putative packaging signals are critical for the influenza life cycle.     

NP, PB1, and PB2 viral genes contribute to altered replication of H5N1 avian influenza viruses in chickens

The virulence determinants for highly pathogenic avian influenza viruses (AIVs) are considered multigenic, although the best characterized virulence factor is the hemagglutinin (HA) cleavage site. The capability of influenza viruses to reassort gene segments is one potential way for new viruses to emerge with different virulence characteristics. To evaluate the role of other gene segments in virulence, we used reverse genetics to generate two H5N1 recombinant viruses with differing pathogenicity in chickens. Single-gene reassortants were used to determine which viral genes contribute to the altered virulence. Exchange of the PB1, PB2, and NP genes impacted replication of the reassortant viruses while also affecting the expression of specific host genes. Disruption of the parental virus' functional polymerase complexes by exchanging PB1 or PB2 genes decreased viral replication in tissues and consequently the pathogenicity of the viruses. In contrast, exchanging the NP gene greatly increased viral replication and expanded tissue tropism, thus resulting in decreased mean death times. Infection with the NP reassortant virus also resulted in the upregulation of gamma interferon and inducible nitric oxide synthase gene expression. In addition to the impact of PB1, PB2, and NP on viral replication, the HA, NS, and M genes also contributed to the pathogenesis of the reassortant viruses. While the pathogenesis of AIVs in chickens is clearly dependent on the interaction of multiple gene products, we have shown that single-gene reassortment events are sufficient to alter the virulence of AIVs in chickens.     

PB1 domains of MEKK2 and MEKK3 interact with the MEK5 PB1 domain for activation of the ERK5 pathway

MEKK2 and MEKK3 are MAPK kinase kinases that activate the ERK5 pathway by phosphorylating and activating the MAPK kinase, MEK5. Activated MEK5 then phosphorylates and activates ERK5. PB1 domains were first defined in the p67phox and Bem1p proteins and have been shown to mediate protein-protein heterodimerization. A PB1 domain is encoded within the N-terminal portion of MEKK2, MEKK3, and MEK5. Herein, we analyze the functional role of MEKK2, MEKK3, and MEK5 PB1 domains in the ERK5 activation pathway. The PB1 domains of MEKK2 and MEKK3 bind the PB1 domain of MEK5 but do not significantly homo- or heterodimerize with one another in vitro. Furthermore, co-immunoprecipitation of MEKK2 and MEK5 from cell lysates shows that they form a complex in vivo. Deletion or mutation of the MEKK2 PB1 domain abolishes MEKK2-MEK5 complexes, demonstrating that the PB1 domain interaction is required for MEKK2-MEK5 interactions. Expression in cells of the MEKK2 or MEKK3 PB1 domain inhibits ERK5 activation, whereas expression of a mutant MEKK2 unable to bind the MEK5 PB1 domain or expression of the p67phox PB1 domain has no effect on ERK5 activation. These findings demonstrate that the PB1 domain mediates the association of MEKK2 and MEKK3 with MEK5 and that the respective PB1 domains of these kinases are critical for regulation of the ERK5 pathway. The free PB1 domain of MEKK2 or MEKK3 functions effectively to inhibit the ERK5 pathway but not the p38 or JNK pathways, demonstrating the specific and unique requirement of the MEKK2 and MEKK3 PB1 domain in regulating ERK5 activation.     

Cell signaling and function organized by PB1 domain interactions

The PB1-domain-containing proteins p62, aPKC, MEKK2/MEKK3, MEK5, and Par-6 play roles in critical cell processes like osteoclastogenesis, angiogenesis, and early cardiovascular development or cell polarity. PB1 domains are scaffold modules that adopt the topology of ubiquitin-like beta-grasp folds that interact with each other in a front-to-back mode to arrange heterodimers or homo-oligomers. The different PB1 domain adaptors provide specificity for PB1 kinases to ensure the effective transmission of cellular signals. Also, recent data suggest that PB1 domains may serve to orchestrate signaling cascades not involving other PB1 domains, such as the MEK5-ERK5 and p62-ERK1 interactions.     

Structural insight into the essential PB1–PB2 subunit contact of the influenza virus RNA polymerase

Influenza virus RNA‐dependent RNA polymerase is a multi‐functional heterotrimer, which uses a ‘cap‐snatching’ mechanism to produce viral mRNA. Host cell mRNA is cleaved to yield a cap‐bearing oligonucleotide, which can be extended using viral genomic RNA as a template. The cap‐binding and endonuclease activities are only activated once viral genomic RNA is bound. This requires signalling from the RNA‐binding PB1 subunit to the cap‐binding PB2 subunit, and the interface between these two subunits is essential for the polymerase activity. We have defined this interaction surface by protein crystallography and tested the effects of mutating contact residues on the function of the holo‐enzyme. This novel interface is surprisingly small, yet, it has a crucial function in regulating the 250 kDa polymerase complex and is completely conserved among avian and human influenza viruses.     

Impact of amino acid mutations in PB2, PB1-F2, and NS1 on the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses

Here, we assessed the effects of PB1-F2 and NS1 mutations known to increase the pathogenicity of influenza viruses on the replication and pathogenicity in mice of pandemic (H1N1) 2009 influenza viruses. We also characterized viruses possessing a PB1-F2 mutation that was recently identified in pandemic (H1N1) 2009 influenza virus isolates, with and without simultaneous mutations in PB2 and NS1. Our results suggest that some NS1 mutations and the newly identified PB1-F2 mutation have the potential to increase the replication and/or pathogenicity of pandemic (H1N1) 2009 influenza viruses.     

Influenza A Virus PB1-F2 Protein Expression Is Regulated in a Strain-Specific Manner by Sequences Located Downstream of the PB1-F2 Initiation Codon

Translation of influenza A virus PB1-F2 occurs in a second open reading frame (ORF) of the PB1 gene segment. PB1-F2 has been implicated in regulation of polymerase activity, immunopathology, susceptibility to secondary bacterial infection, and induction of apoptosis. Experimental evidence of PB1-F2 molecular function during infection has been collected primarily from human and avian viral isolates. As the 2009 H1N1 (H1N1pdm09) strain highlighted, some swine-derived influenza viruses have the capacity to infect human hosts and emerge as a pandemic. Understanding the impact that virulence factors from swine isolates have on both human and swine health could aid in early identification of viruses with pandemic potential. Studies examining PB1-F2 from swine isolates have focused primarily on H1N1pdm09, which does not encode PB1-F2 but was engineered to carry a full-length PB1-F2 ORF to assess the impact on viral replication and pathogenicity. However, experimental evidence of PB1-F2 protein expression from swine lineage viruses has not been demonstrated. Here, we reveal that during infection, PB1-F2 expression levels are substantially different in swine and human influenza viruses. We provide evidence that PB1-F2 expression is regulated at the translational level, with very low levels of PB1-F2 expression from swine lineage viruses relative to a human isolate PB1-F2. Translational regulation of PB1-F2 expression was partially mapped to two independent regions within the PB1 mRNA, located downstream of the PB1-F2 start site. Our data suggest that carrying a full-length PB1-F2 ORF may not be predictive of PB1-F2 expression in infected cells for all influenza A viruses.     

A型流感病毒PB1-F2蛋白与MOAP-1的相互作用

【目的】探讨A型流感病毒PB1-F2蛋白和人类凋亡调节因子1(MOAP-1)之间的相互作用。【方法】构建pACT2-MOAP-1重组质粒,与pGBKT7-PB1-F2质粒共转化酵母AH109,检测转化菌在四缺培养基的生长情况及β半乳糖苷酶报告基因的活性;利用GST pull-down和免疫共沉淀(Co-IP)技术进一步验证PB1-F2与宿主细胞蛋白MOAP-1的相互作用;通过过表达PB1-F2和MOAP-1,检测PB1-F2对MOAP-1蛋白表达水平的影响。【结果】酵母双杂交结果表明,PB1-F2和MOAP-1可以在酵母细胞内特异性结合。GST pull-down和Co-IP实验也进一步证实了这两种蛋白的相互作用,而且PB1-F2可上调外源MOAP-1的蛋白水平。【结论】流感病毒PB1-F2与MOAP-1存在相互作用,PB1-F2可能通过与MOAP-1的相互作用参与调控细胞生长及凋亡过程。     

PB2-588I Enhances 2009 H1N1 Pandemic Influenza Virus Virulence by Increasing Viral Replication and Exacerbating PB2 Inhibition of Beta Interferon Expression

The 2009 pandemic H1N1 influenza virus (pdm/09) is typically mildly virulent in mice. In a previous study, we identified four novel swine isolates of pdm/09 viruses that exhibited high lethality in mice. Comparing the consensus sequences of the PB2 subunit of human isolates of pdm/09 viruses with those of the four swine isolate viruses revealed one consensus mutation: T588I. In this study, we determined that 588T is an amino acid mutation conserved in pdm/09 viruses that was exceedingly rare in previous human influenza isolates. To investigate whether the PB2 with the T5581 mutation (PB2-T558I) has an effect on the increased pathogenicity, we rescued a variant containing PB2-588I (Mex_PB2-588I) in the pdm/09 virus, A/Mexico/4486/2009(H1N1), referred to as Mex_WT (where WT is wild type), and characterized the variant in vitro and in vivo. The results indicated that the mutation significantly enhanced polymerase activity in mammalian cells, and the variant exhibited increased growth properties and induced significant weight loss in a mouse model compared to the wild type. We determined that the mutation exacerbated PB2 inhibition of mitochondrial antiviral signaling protein (MAVS)-mediated beta interferon (IFN-β) expression, and PB2-588I was observed to bind to MAVS more efficiently than PB2-588T. The variant induced lower levels of host IFN-β expression than the WT strain during infection. These findings indicate that the pdm/09 influenza virus has increased pathogenicity upon the acquisition of the PB2-T588I mutation and highlight the need for the continued surveillance of the genetic variation of molecular markers in influenza viruses because of their potential effects on pathogenicity and threats to human health.     

PKA phosphorylation of p62/SQSTM1 regulates PB1 domain interaction partner binding

p62, also known as SQSTM1, is a multi-domain signalling scaffold protein involved in numerous critical cellular functions such as autophagy, apoptosis and inflammation. Crucial interactions relevant to these functions are mediated by the N-terminal Phox and Bem1p (PB1) domain, which is divided into two interaction surfaces, one of predominantly acidic and one of basic character. Most known interaction partners, including atypical protein kinase C (aPKC), bind to the basic surface, and acidic–basic interactions at this interface also allow for p62 homopolymerisation. We identify here that the coupling of p62 to the cAMP signalling system is conferred by both the direct binding of cAMP degrading phosphodiesterase-4 (PDE4) to the acidic surface of the p62 PB1 domain and the phosphorylation of the basic surface of this domain by cAMP-dependent protein kinase (PKA). Such phosphorylation is a previously unknown means of regulating PB1 domain interaction partnerships by disrupting the interaction of p62 with basic surface binding partners, such as aPKCs, as well as p62 homopolymerisation. Thus, we uncover a new regulatory mechanism that connects cAMP signalling with the p62 multi-domain signalling scaffold and autophagy cargo receptor protein.     

Susceptibility of Pseudomonas aeruginosa veterinary isolates to Pbunavirus PB1-like phages

In recent years, antimicrobial-resistant Pseudomonas aeruginosa strains have increased in the veterinary field. Therefore, phage therapy has received significant attention as an approach for overcoming antimicrobial resistance. In this context, we isolated and characterized four Pseudomonas bacteriophages. Phylogenetic analysis showed that the isolated phages are novel Myoviridae Pbunavirus PB1-like phages with ØR12 belonging to a different clade compared with the other three. These phages had distinct lytic activity against 22 P. aeruginosa veterinary isolates. The phage cocktail composed from the PB1-like phages clearly inhibited the occurrence of the phage-resistant variant, suggesting that these phages could be useful in phage therapy.