Comparative Genomics Reveals Insights into the Divergent Evolution of Astigmatic Mites and Household Pest Adaptations.

Highly diversified astigmatic mites comprise many medically important human household pests such as house dust mites causing ∼1-2% of all allergic diseases globally; however, their evolutionary origin and diverse lifestyles including reversible parasitism have not been illustrated at the genomic level, which hampers allergy prevention and our exploration of these household pests. Using six high-quality assembled and annotated genomes, this study not only refuted the monophyly of mites and ticks, but also thoroughly explored the divergence of Acariformes and the diversification of astigmatic mites. In monophyletic Acariformes, Prostigmata known as notorious plant pests first evolved, and then rapidly evolving Astigmata diverged from soil oribatid mites. Within astigmatic mites, a wide range of gene families rapidly expanded via tandem gene duplications, including ionotropic glutamate receptors, triacylglycerol lipases, serine proteases and UDP glucuronosyltransferases. Gene diversification after tandem duplications provides many genetic resources for adaptation to sensing environmental signals, digestion, and detoxification in rapidly changing household environments. Many gene decay events only occurred in the skin-burrowing parasitic mite Sarcoptes scabiei. Throughout the evolution of Acariformes, massive horizontal gene transfer events occurred in gene families such as UDP glucuronosyltransferases and several important fungal cell wall lytic enzymes, which enable detoxification and digestive functions and provide perfect drug targets for pest control. This comparative study sheds light on the divergent evolution and quick adaptation to human household environments of astigmatic mites and provides insights into the genetic adaptations and even control of human household pests.

Cryo-electron Tomography Reveals the Roles of FliY in Helicobacter pylori Flagellar Motor Assembly.

Helicobacter pylori plays a causative role in gastric diseases. The pathogenicity of H. pylori depends on its ability to colonize the stomach guided by motility. FliY is a unique flagellar motor switch component coexisting with the classical FliG, FliM, and FliN switch proteins in some bacteria and has been shown to be essential for flagellation. However, the functional importance of FliY in H. pylori flagellar motor assembly is not well understood. Here, we applied cryo-electron tomography and subtomogram averaging to analyze the in situ structures of flagellar motors from wild-type strain, fliY-null mutant and complementation mutants expressing the N-terminal or C-terminal domain of FliY. Loss of full-length FliY or its C-terminal domain interrupted the formation of an intact C ring and soluble export apparatus, as well as the hook and flagellar filaments. Complementation with FliY C-terminal domain restored all these missing components of flagellar motor. Taken together, these results provide structural insights into the roles of FliY, especially its C-terminal domain in flagellar motor assembly in H. pylori. IMPORTANCE Helicobacter pylori is the major risk factor related with gastric diseases. Flagellar motor is one of the most important virulence factors in H. pylori. However, the assembly mechanism of H. pylori flagellar motor is not fully understood yet. Previous report mainly described the overall structures of flagellum but had not focused on its specific components. Here, we focus on H. pylori flagellar C-ring protein FliY. We directly visualize the flagellar structures of H. pylori wild-type and FliY N-/C-terminal complementary strains by cryo-electron tomography and subtomogram averaging. Our results show that deletion of FliY or its C-terminal domain causes the loss of C ring, whereas deletion of FliY N-terminal does not affect C-ring assembly and flagellar structures. Our results provide direct evidence that C-ring protein FliY, especially its C-terminal domain, plays an indispensable role in H. pylori motor assembly and flagellar formation. This study will deepen our understanding about H. pylori pathogenesis.

Mining of linear B cell epitopes of SARS-CoV-2 ORF8 protein from COVID-19 patients.

Given the on-going SARS-CoV-2 pandemic, identification of immunogenic targets against the viral protein will provide crucial advances towards the development of sensitive diagnostic tools and vaccination strategies. Our previous study has found that ORF8 protein of SARS-CoV-2 is highly immunogenic and shows high sensitivity in identifying COVID-19 disease. In this study, by employing overlapping linear peptides, we characterized the IgG immunodominant regions on SARS-CoV-2 ORF8 protein that are seropositive in the sera from SARS-CoV-2-infected patients. The major immunogenic epitopes are localized at (1) N-termini alpha helix, (2) the resides spanning beta 2 and 3 sheets, and (3) the loop between beta 4 and 5 sheets. Additionally, hamster model infected by SARS-CoV-2 further validates the seropositivity of the linear epitopes in vivo, demonstrating a potential application of the linear peptide-based immunization strategy. Taken together, identification and validation of these B-cell linear epitopes will provide insights into the design of serological diagnostics and peptide-based vaccination approach against this pandemic virus of high priority.

Structure of mouse cytosolic sulfotransferase SULT2A8 provides insight into sulfonation of 7α-hydroxyl bile acids.

Cytosolic sulfotransferases (SULTs) catalyze the transfer of a sulfonate group from the cofactor 3'-phosphoadenosine 5'-phosphosulfate to a hydroxyl (OH) containing substrate and play a critical role in the homeostasis of endogenous compounds, including hormones, neurotransmitters, and bile acids. In human, SULT2A1 sulfonates the 3-OH of bile acids; however, bile acid metabolism in mouse is dependent on a 7α-OH sulfonating SULT2A8 via unknown molecular mechanisms. In this study, the crystal structure of SULT2A8 in complex with adenosine 3',5'-diphosphate and cholic acid was resolved at a resolution of 2.5 Å. Structural comparison with human SULT2A1 reveals different conformations of substrate binding loops. In addition, SULT2A8 possesses a unique substrate binding mode that positions the target 7α-OH of the bile acid close to the catalytic site. Furthermore, mapping of the critical residues by mutagenesis and enzyme activity assays further highlighted the importance of Lys44 and His48 for enzyme catalysis and Glu237 in loop 3 on substrate binding and stabilization. In addition, limited proteolysis and thermal shift assays suggested that the cofactor and substrates have protective roles in stabilizing SULT2A8 protein. Together, the findings unveil the structural basis of bile acid sulfonation targeting 7α-OH and shed light on the functional diversity of bile acid metabolism across species.

Accurate Diagnosis of COVID-19 by a Novel Immunogenic Secreted SARS-CoV-2 orf8 Protein.

An accurate diagnostic test for early severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is the key weapon to control the coronavirus disease 2019 (COVID-19) pandemic. We previously reported that the SARS-CoV-2 genome contains a unique orf8 accessory gene absent from other human-pathogenic coronaviruses. Here, we characterized the SARS-CoV-2 orf8 as a novel immunogenic secreted protein and utilized it for the accurate diagnosis of COVID-19. Extracellular orf8 protein was detected in cell culture supernatant and in sera of COVID-19 patients. In addition, orf8 was found highly immunogenic in COVID-19 patients, who showed early seropositivity for anti-orf8 IgM, IgG, and IgA. We hypothesize that orf8 secretion during SARS-CoV-2 infection facilitates early mounting of B cell response. The serological test detecting anti-orf8 IgG antibody can be used for the early and accurate diagnosis of COVID-19.IMPORTANCE Current commercially available serological tests for COVID-19 patients are detecting antibodies against SARS-CoV-2 nucleoprotein and spike glycoprotein. The antinucleoprotein and antispike antibodies can be accurately detected in patients during the mid or late stage of infection, and therefore, these assays have not been widely used for early diagnosis of COVID-19. In this study, we characterized the secretory property of a SARS-CoV-2 orf8 protein and proposed that orf8 secretion during infection facilitates early mounting of the B cell response. We demonstrated the presence of anti-orf8 antibodies in both symptomatic and asymptomatic patients during the early stage of infection, while the anti-N antibody is not detected. Our serological test detecting anti-orf8 antibodies may facilitate the development of early and accurate diagnosis for COVID-19.

Crystal Structure of Flagellar Export Chaperone FliS in Complex With Flagellin and HP1076 of Helicobacter pylori.

Functional flagella formation is a widespread virulence factor that plays a critical role in survival and host colonization. Flagellar synthesis is a complex and highly coordinated process. The assembly of the axial structure beyond the cell membrane is mediated by export chaperone proteins that transport their cognate substrates to the export gate complex. The export chaperone FliS interacts with flagellin, the basic component used to construct the filament. Unlike enterobacteria, the gastric pathogen Helicobacter pylori produces two different flagellins, FlaA and FlaB, which exhibit distinct spatial localization patterns in the filament. Previously, we demonstrated a molecular interaction between FliS and an uncharacterized protein, HP1076, in H. pylori. Here, we present the crystal structure of FliS in complex with both the C-terminal D0 domain of FlaB and HP1076. Although this ternary complex reveals that FliS interacts with flagellin using a conserved binding mode demonstrated previously in Aquifex aeolicus, Bacillus subtilis, and Salmonella enterica serovar Typhimurium, the helical conformation of FlaB in this complex was different. Moreover, HP1076 and the D1 domain of flagellin share structural similarity and interact with the same binding interface on FliS. This observation was further validated through competitive pull-down assays and kinetic binding analyses. Interestingly, we did not observe any detrimental flagellation or motility phenotypes in an hp1076-null strain. Our localization studies suggest that HP1076 is a membrane-associated protein with a cellular localization independent of FliS. As HP1076 is uniquely expressed in H. pylori and related species, we propose that this protein may contribute to the divergence of the flagellar system, although its relationship with FliS remains incompletely elucidated.

Cryo-EM structures of Helicobacter pylori vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution.

Human gastric pathogen Helicobacter pylori (H. pylori) is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor secreted by H. pylori and induces multiple cellular responses. Although structural and functional studies of VacA have been extensively performed, the high-resolution structure of a full-length VacA protomer and the molecular basis of its oligomerization are still unknown. Here, we use cryoelectron microscopy to resolve 10 structures of VacA assemblies, including monolayer (hexamer and heptamer) and bilayer (dodecamer, tridecamer, and tetradecamer) oligomers. The models of the 88-kDa full-length VacA protomer derived from the near-atomic resolution maps are highly conserved among different oligomers and show a continuous right-handed ß-helix made up of two domains with extensive domain-domain interactions. The specific interactions between adjacent protomers in the same layer stabilizing the oligomers are well resolved. For double-layer oligomers, we found short- and/or long-range hydrophobic interactions between protomers across the two layers. Our structures and other previous observations lead to a mechanistic model wherein VacA hexamer would correspond to the prepore-forming state, and the N-terminal region of VacA responsible for the membrane insertion would undergo a large conformational change to bring the hydrophobic transmembrane region to the center of the oligomer for the membrane channel formation.

Three SpoA-domain proteins interact in the creation of the flagellar type III secretion system in Helicobacter pylori.

Bacterial flagella are rotary nanomachines that contribute to bacterial fitness in many settings, including host colonization. The flagellar motor relies on the multiprotein flagellar motor-switch complex to govern flagellum formation and rotational direction. Different bacteria exhibit great diversity in their flagellar motors. One such variation is exemplified by the motor-switch apparatus of the gastric pathogen Helicobacter pylori, which carries an extra switch protein, FliY, along with the more typical FliG, FliM, and FliN proteins. All switch proteins are needed for normal flagellation and motility in H. pylori, but the molecular mechanism of their assembly is unknown. To fill this gap, we examined the interactions among these proteins. We found that the C-terminal SpoA domain of FliY (FliYC) is critical to flagellation and forms heterodimeric complexes with the FliN and FliM SpoA domains, which are ß-sheet domains of type III secretion system proteins. Surprisingly, unlike in other flagellar switch systems, neither FliY nor FliN self-associated. The crystal structure of the FliYC-FliNC complex revealed a saddle-shaped structure homologous to the FliN-FliN dimer of Thermotoga maritima, consistent with a FliY-FliN heterodimer forming the functional unit. Analysis of the FliYC-FliNC interface indicated that oppositely charged residues specific to each protein drive heterodimer formation. Moreover, both FliYC-FliMC and FliYC-FliNC associated with the flagellar regulatory protein FliH, explaining their important roles in flagellation. We conclude that H. pylori uses a FliY-FliN heterodimer instead of a homodimer and creates a switch complex with SpoA domains derived from three distinct proteins.

Crystal structure of the FliF-FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS-C ring in the bacterial flagellum.

The bacterial flagellar motor is a self-assembling supramolecular nanodevice. Its spontaneous biosynthesis is initiated by the insertion of the MS ring protein FliF into the inner membrane, followed by attachment of the switch protein FliG. Assembly of this multiprotein complex is tightly regulated to avoid nonspecific aggregation, but the molecular mechanisms governing flagellar assembly are unclear. Here, we present the crystal structure of the cytoplasmic domain of FliF complexed with the N-terminal domain of FliG (FliF C -FliG N ) from the bacterium Helicobacter pylori Within this complex, FliF C interacted with FliG N through extensive hydrophobic contacts similar to those observed in the FliF C -FliG N structure from the thermophile Thermotoga maritima, indicating conservation of the FliF C -FliG N interaction across bacterial species. Analysis of the crystal lattice revealed that the heterodimeric complex packs as a linear superhelix via stacking of the armadillo repeat-like motifs (ARM) of FliG N Notably, this linear helix was similar to that observed for the assembly of the FliG middle domain. We validated the in vivo relevance of the FliG N stacking by complementation studies in Escherichia coli Furthermore, structural comparison with apo FliG from the thermophile Aquifex aeolicus indicated that FliF regulates the conformational transition of FliG and exposes the complementary ARM-like motifs of FliG N , containing conserved hydrophobic residues. FliF apparently both provides a template for FliG polymerization and spatiotemporally controls subunit interactions within FliG. Our findings reveal that a small protein fold can serve as a versatile building block to assemble into a multiprotein machinery of distinct shapes for specific functions.

A putative spermidine synthase interacts with flagellar switch protein FliM and regulates motility in Helicobacter pylori.

The flagellar motor is an important virulence factor in infection by many bacterial pathogens. Motor function can be modulated by chemotactic proteins and recently appreciated proteins that are not part of the flagellar or chemotaxis systems. How these latter proteins affect flagellar activity is not fully understood. Here, we identified spermidine synthase SpeE as an interacting partner of switch protein FliM in Helicobacter pylori using pull-down assay and mass spectrometry. To understand how SpeE contributes to flagellar motility, a speE-null mutant was generated and its motility behavior was evaluated. We found that deletion of SpeE did not affect flagellar formation, but induced clockwise rotation bias. We further determined the crystal structure of the FliM-SpeE complex at 2.7 Å resolution. SpeE dimer binds to FliM with micromolar binding affinity, and their interaction is mediated through the ß1' and ß2' region of FliM middle domain. The FliM-SpeE binding interface partially overlaps with the FliM surface that interacts with FliG and is essential for proper flagellar rotational switching. By a combination of protein sequence conservation analysis and pull-down assays using FliM and SpeE orthologues in E. coli, our data suggest that FliM-SpeE association is unique to Helicobacter species.

Degradation of nuclear Ubc9 induced by listeriolysin O is dependent on K+ efflux.

Listeriolysin O (LLO) is a pore-forming toxin produced by L. monocytogenes, and is belonged to a protein family of cholesterol-dependent cytolysins (CDCs). Previous studies have demonstrated that LLO triggers Ubc9 degradation and disrupts host SUMOylation to facilitate bacterial infection. However, the underlying mechanism of Ubc9 degradation is unclear. Here we show that LLO-induced down-regulation of Ubc9 is independent of Ubc9-SUMO interaction, however, it may involve phosphorylation signaling. Additionally, LLO exerts its effects primarily on nuclear Ubc9 and this process is mediated by K+ efflux. Interestingly, for intracellular CDCs such as pneumolysin and suilysin, blockage of K+ efflux enhances degradation of nuclear Ubc9, suggesting that extracellular and intracellular pathogens may exploit different mechanisms to modulate host SUMOylation system. Furthermore, up-regulation of SUMOylation by stable expression of SUMO-1 or SUMO-2 shows a delay in membrane perforation by LLO, indicating that SUMO modification of host proteins may act at the frontline for the defense response against LLO. Taken together, our study provides insights to the understanding of host-pathogen interactions.

Structural basis of the PE-PPE protein interaction in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, has developed multiple strategies to adapt to the human host. The five type VII secretion systems, ESX-1-5, direct the export of many virulence-promoting protein effectors across the complex mycobacterial cell wall. One class of ESX substrates is the PE-PPE family of proteins, which is unique to mycobacteria and essential for infection, antigenic variation, and host-pathogen interactions. The genome of Mtb encodes 168 PE-PPE proteins. Many of them are thought to be secreted through ESX-5 secretion system and to function in pairs. However, understanding of the specific pairing of PE-PPE proteins and their structure-function relationship is limited by the challenging purification of many PE-PPE proteins, and our knowledge of the PE-PPE interactions therefore has been restricted to the PE25-PPE41 pair and its complex with the ESX-5 secretion system chaperone EspG5. Here, we report the crystal structure of a new PE-PPE pair, PE8-PPE15, in complex with EspG5. Our structure revealed that the EspG5-binding sites on PPE15 are relatively conserved among Mtb PPE proteins, suggesting that EspG5-PPE15 represents a more typical model for EspG5-PPE interactions than EspG5-PPE41. A structural comparison with the PE25-PPE41 complex disclosed conformational changes in the four-helix bundle structure and a unique binding mode in the PE8-PPE15 pair. Moreover, homology-modeling and mutagenesis studies further delineated the molecular determinants of the specific PE-PPE interactions. These findings help develop an atomic algorithm of ESX-5 substrate recognition and PE-PPE pairing.

PACT Facilitates RNA-Induced Activation of MDA5 by Promoting MDA5 Oligomerization.

MDA5 is a RIG-I-like cytoplasmic sensor of dsRNA and certain RNA viruses, such as encephalomyocarditis virus, for the initiation of the IFN signaling cascade in the innate antiviral response. The affinity of MDA5 toward dsRNA is low, and its activity becomes optimal in the presence of unknown cellular coactivators. In this article, we report an essential coactivator function of dsRNA-binding protein PACT in mediating the MDA5-dependent type I IFN response. Virus-induced and polyinosinic-polycytidylic acid-induced activation of MDA5 were severely impaired in PACT-knockout cells and attenuated in PACT-knockdown cells, but they were potentiated when PACT was overexpressed. PACT augmented IRF3-dependent type I IFN production subsequent to dsRNA-induced activation of MDA5. In contrast, PACT had no influence on MDA5-mediated activation of NF-κB. PACT required dsRNA interaction for its action on MDA5 and promoted dsRNA-induced oligomerization of MDA5. PACT had little stimulatory effect on MDA5 mutants deficient for oligomerization and filament assembly. PACT colocalized with MDA5 in the cytoplasm and potentiated MDA5 recruitment to the dsRNA ligand. Taken together, these findings suggest that PACT functions as an essential cellular coactivator of RIG-I, as well as MDA5, and it facilitates RNA-induced formation of MDA5 oligomers.

Helicobacter pylori does not use spermidine synthase to produce spermidine.

Helicobacter pylori is the primary pathogen associated to gastritis and gastric cancer. Growth of H. pylori depends on the availability of spermidine in vivo. Interestingly, the genome of H. pylori contains an incomplete set of genes for the classical pathway of spermidine biosynthesis. It is thus not clear whether some other genes remained in the pathway would have any functions in spermidine biosynthesis. Here, we study spermidine synthase, which is responsible for the final catalytic process in the classical route. Protein sequence alignment reveals that H. pylori SpeE (HpSpeE) lacks key residues for substrate binding. By using isothermal titration calorimetry, we show that purified recombinant HpSpeE does not interact with the putative substrates putrescine and decarboxylated S-adenosylmethionine, and the product spermidine. High performance liquid chromatography analysis further demonstrates that HpSpeE has no detectable in vitro enzymatic activity. Additionally, intracellular spermidine level in speE-null mutant strain is comparable to that in the wild type strain. Collectively, our results suggest that HpSpeE is functionally distinct from spermidine production. H. pylori may instead employ the alternative pathway for spermidine synthesis which is dominantly exploited by other human gut microbes.

Suppression of PACT-induced type I interferon production by herpes simplex virus 1 Us11 protein.

Herpes simplex virus 1 (HSV-1) Us11 protein is a double-stranded RNA-binding protein that suppresses type I interferon production through the inhibition of the cytoplasmic RNA sensor RIG-I. Whether additional cellular mediators are involved in this suppression remains to be determined. In this study, we report on the requirement of cellular double-stranded RNA-binding protein PACT for Us11-mediated perturbation of type I interferon production. Us11 associates with PACT tightly to prevent it from binding with and activating RIG-I. The Us11-deficient HSV-1 was indistinguishable from the Us11-proficient virus in the suppression of interferon production when PACT was compromised. More importantly, HSV-1-induced activation of interferon production was abrogated in PACT knockout murine embryonic fibroblasts. Our findings suggest a new mechanism for viral evasion of innate immunity through which a viral double-stranded RNA-binding protein interacts with PACT to circumvent type I interferon production. This mechanism might also be used by other PACT-binding viral interferon-antagonizing proteins such as Ebola virus VP35 and influenza A virus NS1.

Structural basis of FliG-FliM interaction in Helicobacter pylori.

FliG and FliM are switch proteins that regulate the rotation and switching of the flagellar motor. Several assembly models for FliG and FliM have recently been proposed; however, it remains unclear whether the assembly of the switch proteins is conserved among different bacterial species. We applied a combination of pull-down, thermodynamic and structural analyses to characterize the FliM-FliG association from the mesophilic bacterium Helicobacter pylori. FliM binds to FliG with micromolar binding affinity, and their interaction is mediated through the middle domain of FliG (FliGM ), which contains the EHPQR motif. Crystal structures of the middle domain of H. pylori FliM (FliM(M)) and FliG(M) -FliM(M) complex revealed that FliG binding triggered a conformational change of the FliM α3-α1' loop, especially Asp130 and Arg144. We furthermore showed that various highly conserved residues in this region are required for FliM-FliG complex formation. Although the FliM-FliG complex structure displayed a conserved binding mode when compared with Thermotoga maritima, variable residues were identified that may contribute to differential binding affinities across bacterial species. Comparison of the thermodynamic parameters of FliG-FliM interactions between H. pylori and Escherichia coli suggests that molecular basis and binding properties of FliM to FliG is likely different between these two species.

Genetically encodable design of ligand "bundling" on the surface of nanoparticles.

Polyhistidine peptide dendrimer self-assembles on CdSe/ZnS quantum dots (QDs) with very high affinity and stability, a property ascribable to its multivalent geometry. Here we designed a fluorescent protein, GCN-mCherry, that exists as an oligomeric bundled structure in solution as well as on the surface to imitate the structure of a synthetic dendrimer. GCN-mCherry forms a very stable assembly with QDs, which can resist displacement by 500 mM imidazole and the dendrimer peptide, as measured by the Förster resonance energy transfer from QD to mCherry. Our work manifested a prominent stability enhancement of protein-nanoparticle assembly through directional ligand-ligand interaction on the surface.

Influenza polymerase activity correlates with the strength of interaction between nucleoprotein and PB2 through the host-specific residue K/E627.

The ribonucleoprotein (RNP) complex is the essential transcription-replication machinery of the influenza virus. It is composed of the trimeric polymerase (PA, PB1 and PB2), nucleoprotein (NP) and RNA. Elucidating the molecular mechanisms of RNP assembly is central to our understanding of the control of viral transcription and replication and the dependence of these processes on the host cell. In this report, we show, by RNP reconstitution assays and co-immunoprecipitation, that the interaction between NP and polymerase is crucial for the function of the RNP. The functional association of NP and polymerase involves the C-terminal '627' domain of PB2 and it requires NP arginine-150 and either lysine-627 or arginine-630 of PB2. Using surface plasmon resonance, we demonstrate that the interaction between NP and PB2 takes place without the involvement of RNA. At 33, 37 and 41°C in mammalian cells, more positive charges at aa. 627 and 630 of PB2 lead to stronger NP-polymerase interaction, which directly correlates with the higher RNP activity. In conclusion, our study provides new information on the NP-PB2 interaction and shows that the strength of NP-polymerase interaction and the resulting RNP activity are promoted by the positive charges at aa. 627 and 630 of PB2.

Structural basis for RNA binding and homo-oligomer formation by influenza B virus nucleoprotein.

Influenza virus nucleoprotein (NP) is the major component of the viral ribonucleoprotein complex, which is crucial for the transcription and replication of the viral genome. We have determined the crystal structure of influenza B virus NP to a resolution of 3.2 Å. Influenza B NP contains a head, a body domain, and a tail loop. The electropositive groove between the head and body domains of influenza B NP is crucial for RNA binding. This groove also contains an extended flexible charged loop (amino acids [aa] 125 to 149), and two lysine clusters at the first half of this loop were shown to be crucial for binding RNA. Influenza B virus NP forms a crystallographic homotetramer by inserting the tail loop into the body domain of the neighboring NP molecule. A deeply buried salt bridge between R472 and E395 and a hydrophobic cluster at F468 are the major driving forces for the insertion. The analysis of the influenza B virus NP structure and function and comparisons with influenza A virus NP provide insights into the mechanisms of action and underpin efforts to design inhibitors for this class of proteins.

Multiple conformations of the FliG C-terminal domain provide insight into flagellar motor switching.

Bacterial flagellar switching between counterclockwise and clockwise directions is mediated by the coupling of the chemotactic system and the motor switch complex. The conformational changes of FliG are closely associated with this switching mechanism. We present two crystal structures of FliG(MC) from Helicobacter pylori, each showing distinct domain orientations from previously solved structures. A 180° rotation of the charged ridge-containing C-terminal subdomain FliG(Cα1-6) that is prompted by the rotational freedom of Met245 psi and Phe246 phi at the MFXF motif was revealed. Studies on the swarming and swimming behavior of Escherichia coli mutants further identified the importance of the 245MFXF248 motif and a highly conserved residue, Asn216, in motor switching. Additionally, multiple conformations of FliG(Cα1-6) were demonstrated by intramolecular cysteine crosslinking. The conformational flexibility of FliGc leads us to propose a model that accounts for the symmetrical torque generation process and for the dynamics of the motor.