CD Loop Research Articles

Microcrystal electron diffraction structure of Toll-like receptor 2 TIR-domain-nucleated MyD88 TIR-domain higher-order assembly.

Eukaryotic TIR (Toll/interleukin-1 receptor protein) domains signal via TIR-TIR interactions, either by self-association or by interaction with other TIR domains. In mammals, TIR domains are found in Toll-like receptors (TLRs) and cytoplasmic adaptor proteins involved in pro-inflammatory signaling. Previous work revealed that the MAL TIR domain (MALTIR) nucleates the assembly of MyD88TIR into crystalline arrays in vitro. A microcrystal electron diffraction (MicroED) structure of the MyD88TIR assembly has previously been solved, revealing a two-stranded higher-order assembly of TIR domains. In this work, it is demonstrated that the TIR domain of TLR2, which is reported to signal as a heterodimer with either TLR1 or TLR6, induces the formation of crystalline higher-order assemblies of MyD88TIR in vitro, whereas TLR1TIR and TLR6TIR do not. Using an improved data-collection protocol, the MicroED structure of TLR2TIR-induced MyD88TIR microcrystals was determined at a higher resolution (2.85 Å) and with higher completeness (89%) compared with the previous structure of the MALTIR-induced MyD88TIR assembly. Both assemblies exhibit conformational differences in several areas that are important for signaling (for example the BB loop and CD loop) compared with their monomeric structures. These data suggest that TLR2TIR and MALTIR interact with MyD88 in an analogous manner during signaling, nucleating MyD88TIR assemblies unidirectionally.

Cd loop fusion enhances the immunogenicity and the potential transmission blocking activity of Plasmodium falciparum generative cell specific 1 (GCS1) antigen

TBVs are suggested to inhibit parasite transmission from humans to Anopheles mosquitoes. For the transmission of Plasmodium parasite, a variety of factors are included in gametes fusion phase. In this step, conserved male-specific generative cell specific 1 antigen is necessary for fusion of cytoplasmic membranes of micro- and macro-gametocytes and zygot formation. The partial blocking activities of elicited antibodies against either the HAP2-GCS1 domain or the cd loop of this antigen have been recorded to hinder the transmission of Plasmodium species in Anopheles mid-gut. Thus, the objective of the present study was to investigate if the cd loop-fusion can enhance the quantity and quality of humoral and cellular immune responses against Plasmodium falciparum GCS1 in comparison to non-fusion antigen (without cd loop), in the adjuvanted and non-adjuvanted mouse groups. The immunogenicity of two constructs of P. falciparum generative cell specific 1 antigen, a fusion protein composed of cd loop and HAP2-GCS1 domain (cd-HAP) and another recombinant PfGCS1 containing solo HAP2-GCS1 domain (HAP2) were assessed to impede Plasmodium gametocytes integration before zygote formation. The antibodies profiling, titer, and avidity of induced antibodies were measured by the immunized mice sera, and the released cytokines (IL-5, TNF, and INF-γ) were analyzed in the supernatants of stimulated splenocytes. Furthermore, the inhibitory potency of the elicited antibodies against HAP2 and cd-HAP was measured during oocyst development by Standard Membrane Feeding Assay (SMFA). The comparative results in the present study showed the higher titer of IgG antibodies and IgG2a subclass, avidity, and transmission-reducing activity (TRA = 72.5 %) when mice were immunized by cd-HAP rather than HAP2. Moreover, our findings confirmed intensified Th1-directed immune responses in group 4 received cd-HAP/Poly(I:C). These findings declared the potential ability of cd loop fusion (cd-HAP) to upsurge humoral and cellular immune responses. However, the immune responses may switch to stronger Th1-type using alternative formulations. Explicitly, the cd-HAP-based vaccine may enhance the overall efficiency of immune responses and present a promising implementation in aiming malaria transmission.

The Structural Framework and Opening Appearance of the VP1-Pocket of Enteroviruses Correlated with Viral Thermostability.

Enteroviruses (EVs and RVs) are prevalent worldwide and cause various diseases in humans, of which the VP1-pocket is a target of antivirals, with a lipid molecule as a pocket factor to stabilize the virion. However, the characterization of the structure of the VP1-pocket in EVs is poor. Here, we compared the published capsid crystals of EVs and RVs and proposed a structural framework for the VP1-pocket: Frame 1-4, which is located at the CD loop, GH loop, and C-terminus, presenting with an outward opening appearance or not. The non-outward viral strains-CVB3, Echo 11, RV-A81, and RV-B70-are more thermally stable, with a breakpoint temperature (B.T.) of 51~62 °C for genome releasing, which is 4~10 °C higher than its outward temperature of 41~47 °C, and infectivity preservation when treated at 50 °C for 3 min. Its outward versus non-outward opening is correlated significantly with the B.T. for genome release (r = -0.90; p = 0.0004) and infectivity (r = -0.82, p = 0.0039). The energy of Frames 1, 2, and 4, including Van der Waals attractive and repulsive interactions and hydrogen bonds, showed significant correlations with the B.T. (r = -0.67, 0.75, and -0.8; p = 0.034, 0.013, and 0.006, respectively). These characters of the VP1-pocket could be predictors for virion thermostability and aid in the development of vaccines or antivirals.

Investigation of the Solid-State Interactions in Lyophilized Human G-CSF Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating factor (G-CSF) formulations, confirming predictions made using computational structure docking. More recently, solid-state HDX mass spectrometry (ssHDX-MS) was developed for proteins in the lyophilized state. Deuterium uptake in ssHDX-MS has been shown for various proteins, including monoclonal antibodies, to be highly correlated with storage stability, as measured by protein aggregation and chemical degradation. As G-CSF is known to lose activity through aggregation upon lyophilization, we applied the ssHDX-MS method with peptide mapping to four different lyophilized formulations of G-CSF to compare the impact of three excipients on local structure and exchange dynamics. HDX at 22 °C was confirmed to correlate well with the monomer content remaining after lyophilization and storage at -20 °C, with sucrose providing the greatest protection, and then phenylalanine, mannitol, and no excipient leading to progressively less protection. Storage at 45 °C led to little difference in final monomer content among the formulations, and so there was no discernible relationship with total deuterium uptake on ssHDX. Incubation at 45 °C may have led to a structural conformation and/or aggregation mechanism no longer probed by HDX at 22 °C. Such a conformational change was observed previously at 37 °C for liquid-formulated G-CSF using NMR. Peptide mapping revealed that tolerance to lyophilization and -20 °C storage was linked to increased stability in the small helix, loop AB, helix C, and loop CD. LC-MS HDX and NMR had previously linked loop AB and loop CD to the formation of a native-like state (N*) prior to aggregation in liquid formulations, suggesting a similar structural basis for G-CSF aggregation in the liquid and solid states.

Molecular Insights into O-Linked Sialoglycans Recognition by the Siglec-Like SLBR-N (SLBRUB10712) of Streptococcus gordonii.

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3'SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections.

Optimized Refolding Buffers Oriented Humoral Immune Responses Versus PfGCS1 Self-Assembled Peptide Nanoparticle.

Developing a novel class of vaccine is pivotal for eliminating and eradicating malaria. Preceding investigations demonstrated partial blocking activity in malaria transmission against recombinant vaccine PfHAP2-GCS1 and conserved region of the cd loop. The effectiveness of immune response varies with the size and shape of the self-assembly of peptide nanoparticles (SAPNs) displaying antigen, affected by different components in refolding buffers. Plasmodium falciparum Generative Cell Specific 1 (PfGCS1), a promising malaria transmission-blocking vaccine (TBV) candidate, was expressed, purified, and followed by a four-step refolding process to form nanoparticles (PfGCS1-SAPNs). The influence of buffer components on the size and shape of SAPNs was investigated by DLS and FESEM. Furthermore, the immunogenicity of nanostructures was assessed in different mouse groups. The results showed that PfGCS1-SAPN was immunogenic and its administration with Poly (I:C), stimulated humoral and cellular responses in the mouse model. In the immunized mice groups, the level of IgG antibodies against PfGCS1-SAPN was significantly increased in different time points (second and third boost) and heterogeneous boosters. The various IgG-subclasses profile shifted to Th1, Th2, or Th1/Th2 mix responses in mice immunized with PfGCS1-SAPN refolded in different buffers, indicating a prerequisite for further investigations to optimize vaccine formulation to enhance and modulate Th1/cellular responses. Such studies pave the way to improve biophysical features related to the nanoparticles' size, shape, and conformational epitopes of candidate antigens and T- and B-cells presented on the superficial structure to elicit robust immune responses.

The circularly permuted globin domain of androglobin exhibits atypical heme stabilization and nitric oxide interaction.

In the decade since the discovery of androglobin, a multi-domain hemoglobin of metazoans associated with ciliogenesis and spermatogenesis, there has been little advance in the knowledge of the biochemical and structural properties of this unusual member of the hemoglobin superfamily. Using a method for aligning remote homologues, coupled with molecular modelling and molecular dynamics, we have identified a novel structural alignment to other hemoglobins. This has led to the first stable recombinant expression and characterization of the circularly permuted globin domain. Exceptional for eukaryotic globins is that a tyrosine takes the place of the highly conserved phenylalanine in the CD1 position, a critical point in stabilizing the heme. A disulfide bond, similar to that found in neuroglobin, forms a closed loop around the heme pocket, taking the place of androglobin's missing CD loop and further supporting the heme pocket structure. Highly unusual in the globin superfamily is that the heme iron binds nitric oxide as a five-coordinate complex similar to other heme proteins that have nitric oxide storage functions. With rapid autoxidation and high nitrite reductase activity, the globin appears to be more tailored toward nitric oxide homeostasis or buffering. The use of our multi-template profile alignment method to yield the first biochemical characterisation of the circularly permuted globin domain of androglobin expands our knowledge of the fundamental functioning of this elusive protein and provides a pathway to better define the link between the biochemical traits of androglobin with proposed physiological functions.

Evaluation of a new fusion antigen, cd loop and HAP2-GCS1 domain (cd-HAP) of Plasmodium falciparum Generative Cell Specific 1 antigen formulated with various adjuvants, as a transmission blocking vaccine

BackgroundMalaria is a major global health challenge, and for the elimination and eradication of this disease, transmission-blocking vaccines (TBVs) are a priority. Plasmodium falciparum Generative Cell Specific 1 (PfGCS1), a promising TBV candidate, is essential for gamete fertilization. The HAP2-GCS1 domain of this antigen as well as its cd loop could induce antibodies that partially inhibit transmission of P. falciparum.MethodsIn the current study, a new synthetic fusion antigen containing cd loop and HAP2-GCS1 domain (cd-HAP) of PfGCS1 was evaluated as a transmission blocking vaccine candidate. Initially, the profile of naturally acquired IgG antibodies to the cd-HAP antigen was analysed in Iranian individuals infected with P. falciparum, to confirm that this new fusion protein has the appropriate structure containing common epitopes with the native form of PfGCS1. Then, the immunogenicity of cd-HAP was evaluated in BALB/c mice, using different adjuvant systems such as CpG, MPL, QS-21, and a combination of them (CMQ). Furthermore, the blocking efficacy of polyclonal antibodies induced against these formulations was also assessed by oocyst intensity and infection prevalence in the Standard Membrane Feeding Assay (SMFA).ResultsThe naturally acquired antibodies (dominantly IgG1 and IgG3 subclasses) induced in P. falciparum-infected individuals could recognize the cd-HAP antigen which implies that the new fusion protein has a proper conformation that mimics the native structure of PfGCS1. Concerning the immunogenicity of cd-HAP antigen, the highest IgG levels and titers, by a Th1-type immune profile, and elevated antibody avidity were induced in mice immunized with the cd-HAP antigen formulated with a combination of adjuvants (P < 0.0001). Additionally, cytokine profiling of the immunized mice displayed that a high level of IFN-γ response, a Th1-type immune response, was produced by splenocytes from immunized mice that received cd-HAP antigen in combination with CMQ adjuvants (P < 0.0001). This formulation of cd-HAP antigen with CMQ adjuvants could reduce oocyst intensity and infection prevalence by 82%, evidenced by the SMFA and hold significant implications for future malaria vaccine development.ConclusionAltogether, the results showed that cd-HAP antigen formulated with a combination of the adjuvants (CMQ), could be a promising formulation to develop a PfGCS1-based transmission-blocking vaccine.

IL-2 Flexible Loops Might Play a Role in IL-2 Interaction with the High-Affinity IL-2 Receptor: A Molecular Dynamics (MD) Study

The clinical use of high-dose IL-2 in cancer immunotherapy faces several drawbacks such as toxicity and unfavorable pharmacokinetic profile. These drawbacks can be avoided by inhibiting IL-2 interaction with the CD25 subunit, which is a component of the high-affinity IL-2 receptor (IL-2Rαβγ). Several studies showed mutations of potential IL-2 residues such as R38, F42, Y45, and Y72 would produce IL-2 that is CD25-independent. In essence, structural comparison between wild-type (WT) IL-2 and CD25-independent IL-2 can be very insightful to assess the role of IL-2 flexibility and conformation in the IL-2 receptor interactions. Here, we investigated the flexibility loops and conformation of IL-2m (F24A, Y45A, and L72G), which is known to be CD25-independent, and IL-2m2 (F42Y and L72R) mutants along with WT IL-2 using MD simulations. Despite residue mutations, both IL-2m and IL-2m2 showed comparable conformational compactness and better stability than WT IL-2. Interestingly, IL-2m and IL-2m2 mutants showed rigid BC and CD loops in comparison to WT IL-2 . Also, the AB loop conformation of IL-2m was a bent structure compared to the WT IL-2 and IL-2m2. Principal component analysis (PCA) and free-energy landscape results suggested IL-2m and IL-2m2 have stable conformations compared to the WT IL-2. Therefore, these mutation sites of IL-2 produced stable and rigid loops that might prevent IL-2 from binding to the CD25 subunit. Our results can help to assess IL-2 flexibility loops to design new CD25-independent IL-2 mutants without compromising the IL-2 structure.

Toxic Misfolded Transthyretin Oligomers with Different Molecular Conformations Formed through Distinct Oligomerization Pathways.

Protein aggregation is initiated by structural changes from native polypeptides to cytotoxic oligomers, which form cross-β structured amyloid. Identification and characterization of oligomeric intermediates are critically important for understanding not only the molecular mechanism of aggregation but also the cytotoxic nature of amyloid oligomers. Preparation of misfolded oligomers for structural characterization is, however, challenging because of their transient, heterogeneous nature. Here, we report two distinct misfolded transthyretin (TTR) oligomers formed through different oligomerization pathways. A pathogenic TTR variant with a strong aggregation propensity (L55P) was used to prepare misfolded oligomers at physiological pH. Our mechanistic studies showed that the full-length TTR initially forms small oligomers, which self-assemble into short protofibrils at later stages. Enzymatic cleavage of the CD loop was also used to induce the formation of N-terminally truncated oligomers, which was detected in ex vivo cardiac TTR aggregates extracted from the tissues of patients. Structural characterization of the oligomers using solid-state nuclear magnetic resonance and circular dichroism revealed that the two TTR misfolded oligomers have distinct molecular conformations. In addition, the proteolytically cleaved TTR oligomers exhibit a higher surface hydrophobicity, suggesting the presence of distinct oligomerization pathways for TTR oligomer formation. Cytotoxicity assays also revealed that the cytotoxicity of cleaved oligomers is stronger than that of the full-length TTR oligomers, indicating that hydrophobicity might be an important property of toxic oligomers. These comparative biophysical analyses suggest that the toxic cleaved TTR oligomers formed through a different misfoling pathway may adopt distinct structural features that produce higher surface hydrophobicity, leading to the stronger cytotoxic activities.

The HIV-1 protein Nef activates the Tec family kinase Btk by stabilizing an intermolecular SH3-SH2 domain interaction.

The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms. The Btk SH3-SH2 region formed a homodimer requiring the CD loop in the SH2 domain, which was stabilized by the binding of Nef homodimers. Alanine substitution of Pro327 in the CD loop of the Btk SH2 domain destabilized SH3-SH2 dimers, abolished the interaction with Nef, and prevented activation by Nef in vitro. In cells, Nef stabilized and activated wild-type but not P327A Btk homodimers at the plasma membrane. These data reveal that the interaction with Nef stabilizes Btk dimers through the SH3-SH2 interface to promote kinase activity and show that the HIV-1 Nef protein evolved distinct mechanisms to activate Src and Tec family tyrosine kinases to enhance viral replication.

Design and development of a self-assembling protein nanoparticle displaying PfHAP2 antigenic determinants recognized by natural acquired antibodies.

BackgroundsIn order to move towards the elimination and eradication of malaria in the world, the development of vaccines is inevitable. Many modern vaccines are based on recombinant technology; however, they may not provide a fully protective, long-lasting immune response. One of the strategies to improve recombinant vaccines is designing the nanovaccines such as self-assembling protein nanoparticles (SAPNs). Hence, the presentation of epitopes in a repeat array and correct conformation should be considered. P. falciparum generative cell-specific 1 (PfGCS1) is a main transmission-blocking vaccine candidate with two highly conserved fragments, HAP2-GCS1 and cd loop, inducing partial malaria transmission inhibitory antibodies. Therefore, to design an effective malaria vaccine, we used cd loop and HAP2-GCS1 fragments at the amino and carboxy terminuses of the SAPN-forming amino acid sequence, respectively.Methodology/Principal findingsThe SAPN monomer (PfGCS1-SAPN) sequence was designed, and the three-dimensional (3D) structure was predicted. The result of this prediction ensured the presence of antigens on the SAPN surface. Then the accuracy of the predicted 3D structure and its stability were confirmed by 100 ns molecular dynamics (MD) simulation. The designed SAPN substructure sequence was synthesized, cloned, and expressed in Escherichia coli. With a gradual decrease in urea concentration in dialysis solutions, the purified proteins progressed to the final desired structure of the SAPN, which then was confirmed by Dynamic Light Scattering (DLS) and Field Emission Scanning Electron Microscopy (FESEM) tests. According to the Enzyme-Linked Immunosorbent Assay (ELISA), antigenic determinants were presented on the SAPN surface and interacted with antibodies in the serum of malaria patients.Conclusions/SignificanceOur results show that the SAPN formed by PfGCS1-SAPN has produced the correct shape and size, and the antigenic determinants are presented on the surface of the SAPN, which indicates that the designed SAPN has great potential to be used in the future as a malaria vaccine.

Molecular genotypic analysis of porcine circovirus type 2 reveals the predominance of PCV2d in Vietnam (2018-2020) and the association between PCV2h, the recombinant forms, and Vietnamese vaccines.

We conducted nucleotide and amino acid sequence alignment and phylogenetic analysis of porcine circovirus ORF2 (Cap protein) from 17 PCV2-positive clinical samples from nine different northern Vietnamese provinces (Mar 2018-Nov 2020), four local vaccines, and 77 reference strains. We identified one PCV2a (1/17 = 5.9%), five PCV2b (5/17 = 29.9%), and 11 PCV2d (11/17 = 64.7%) isolates, while only PCV2d was detected in 2020. Timeline analysis indicated an increasing predominance of PCV2d nationwide (2018-2020). With strong nodal support (98% for nucleotides and 74% for amino acids), the phylogenetic tree topology revealed a distinct PCV2h clade including recombinant/intermediate strains and local vaccines. The Cap protein sequences from 11 PCV2d field strains had the 2d-genotype-typical motif 86SNPLSV91 in loop CD, the motif TGID in loop GH-HI, and the motif 230PLNPK234 in loop CT. The PCV2h isolates (and vaccines) had the 86SNPLSV91, SAID, and 230L(N/H)PK234 motifs. Selection pressure analysis indicated positive selection at seven sites: A68N in immunoreactive region (IRR)-A; 119G and 130V in IRR-B; and 167L, T190(A/S), 194D and 202F in IRR-C. We identified PCV2h as the genotype of the recombinant strains, which resulted from intergenotype recombination of PCV2a, PCV2b, and PCV2d. The current data provide new information about the diversity, distribution, and dominance of the PCV2 genotype in Vietnam.

Protein Engineering and HDX Identify Structural Regions of G-CSF Critical to Its Stability and Aggregation.

The protein engineering and formulation of therapeutic proteins for prolonged shelf-life remain a major challenge in the biopharmaceutical industry. Understanding the influence of mutations and formulations on the protein structure and dynamics could lead to more predictive approaches to their improvement. Previous intrinsic fluorescence analysis of the chemically denatured granulocyte colony-stimulating factor (G-CSF) suggested that loop AB could subtly reorganize to form an aggregation-prone intermediate state. Hydrogen deuterium exchange mass spectrometry (HDX-MS) has also revealed that excipient binding increased the thermal unfolding transition midpoint (Tm) by stabilizing loop AB. Here, we have combined protein engineering with biophysical analyses and HDX-MS to reveal that increased exchange in a core region of the G-CSF comprising loop AB (ABI, a small helix, ABII) and loop CD packed onto helix B and the beginning of loop BC leads to a decrease in Tm and higher aggregation rates. Furthermore, some mutations can increase the population of the aggregation-prone conformation within the native ensemble, as measured by the greater local exchange within this core region.

Structure, Antigenic Properties, and Highly Efficient Assembly of PCV4 Capsid Protein.

Porcine circovirus type 4 (PCV4), a recently reported circovirus, was first identified in pigs with clinical signs similar to porcine dermatitis nephropathy syndrome (PDNS), in Hunan province, China, in 2019. More knowledge regarding the assembly of capsid protein (Cap) into virus-like particles (VLPs), their structure and antigenic properties, are needed to provide new knowledge for diagnosis and further characterization of PCV4. In this study, high-level expression of PCV4 Cap was achieved in Escherichia coli with purified Cap self-assembling into VLPs (~20 nm) in vitro. Furthermore, these VLPs were internalized in vitro by PK15 and 3D4/21 cell lines. Significant structural differences between PCV4 and PCV2 capsids were demonstrated among loops (loop BC, CD, DE, EF, and GH), based on comparisons of 3D structures. In addition, five potential B cell epitopes identified in silico were mostly located in surface-exposed loops of PCV4 capsid. Cross-reaction between PCV4 and PCV2 or PCV3 conferred by humoral immune responses was deemed unlikely on the basis of ELISA and Western blotting for assessment of VLPs and using PCV4 or PCV2 VLPs. In conclusion, these studies provided new knowledge regarding PCV4 capsid surface patterns. It is noteworthy that the PCV4 VLPs prepared in our study have much potential for development of serological diagnostics for PCV4 and to further characterize this virus.

Displaying epitope B and epitope 7 of porcine reproductive and respiratory syndrome virus on virus like particles of porcine circovirus type 2 provides partial protection to pigs

The Cap of porcine circovirus type 2 (PCV2) can be assembled into virus like particles (VLPs) in vitro that have multiple loops located on the particle surface. This would make it a good vehicle for displaying exogenous proteins or epitopes. We derived two epitopes, epitope B (EpB, S37HIQLIYNL45) and epitope 7 (Ep7, Q196WGRL200) from Gp5 of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). We replaced the core region of Loop CD (L75PPGGGSN82) and the carboxyl terminus (K222DPPL226) of PCV2 Cap, respectively, to construct a bi-epitope chimeric PCV2 Cap. Its immunogenicity and protective effects were evaluated as one PRRSV subunit vaccine. The chimeric PCV2 Cap was soluble, efficiently expressed in an Escherichia coli expression system, and could be self-assembled into chimeric virus like particles (cVLPs) with a diameter of 12–15 nm. Western blotting confirmed that the cVLPs could be specifically recognized by anti-PCV2, anti-EpB and anti-Ep7 antibodies. The cVLPs vaccine could alleviate the clinical symptoms and reduce the viral loads after HP-PRRSV challenge in 100–120 days old pigs. These data suggest that the cVLPs vaccine could provide pigs with partial protection against homologous PRRSV strains, and it provides a new design for additional PRRSV subunit vaccines.

The unique structure of the zebrafish TNF-α homotrimer

In the current study, zebrafish TNF-α1 (zTNF-α1) was crystallized, and the structure was analyzed. The zTNF-α1 trimer is composed of three monomers whose height and width are 50 Å and 60 Å, respectively. Compared with human TNF-α, zTNF-α1 shows only ~30% amino acid identity, the EF loop of each monomer lacks three amino acids, the CD loop is increased by four amino acids, and the AA’’ loop is increased by one amino acid. In addition, an A″-β-chain is added to the zTNF-α1 monomer, forming two β-sheet layers with 6:5 β-chains. The top of the trimer is missing three amino acids and the inner coil because the EF loop seals the central hole at the top, forming a unique structure. In conclusion, the results elucidated the structure of the zTNF-α1 trimer, providing immunological knowledge for studying TNF-α function in the zebrafish animal model and structural information for exploring TNF-α family evolution.

In silico screening and analysis of nonsynonymous SNPs in human CYP1A2 to assess possible associations with pathogenicity and cancer susceptibility

Cytochrome P450 1A2 (CYP1A2) is one of the main hepatic CYPs involved in metabolism of carcinogens and clinically used drugs. Nonsynonymous single nucleotide polymorphisms (nsSNPs) of this enzyme could affect cancer susceptibility and drug efficiency. Hence, identification of human CYP1A2 pathogenic nsSNPs could be of great importance in personalized medicine and pharmacogenetics. Here, 176 nsSNPs of human CYP1A2 were evaluated using a variety of computational tools, of which 18 nsSNPs were found to be associated with pathogenicity. Further analysis suggested possible association of 9 nsSNPs (G73R, G73W, R108Q, R108W, E168K, E346K, R431W, F432S and R456H) with the risk of hepatocellular carcinoma. Molecular dynamics simulations revealed higher overall flexibility, decreased intramolecular hydrogen bonds and lower content of regular secondary structures for both cancer driver variants G73W and F432S when compared to the wild-type structure. In case of F432S, loss of the conserved hydrogen bond between Arg137 and heme propionate oxygen may affect heme stability and the observed significant rise in fluctuation of the CD loop could modify CYP1A2 interactions with its redox partners. Together, these findings propose CYP1A2 as a possible candidate for hepatocellular carcinoma and provide structural insights into how cancer driver nsSNPs could affect protein structure, heme stability and interaction network.

Allostery in the nitric oxide dioxygenase mechanism of flavohemoglobin

The substrates O2 and NO cooperatively activate the NO dioxygenase function of Escherichia coli flavohemoglobin. Steady-state and transient kinetic measurements support a structure-based mechanistic model in which O2 and NO movements and conserved amino acids at the E11, G8, E2, E7, B10, and F7 positions within the globin domain control activation. In the cooperative and allosteric mechanism, O2 migrates to the catalytic heme site via a long hydrophobic tunnel and displaces LeuE11 away from the ferric iron, which forces open a short tunnel to the catalytic site gated by the ValG8/IleE15 pair and LeuE11. NO permeates this tunnel and leverages upon the gating side chains triggering the CD loop to furl, which moves the E and F-helices and switches an electron transfer gate formed by LysF7, GlnE7, and water. This allows FADH2 to reduce the ferric iron, which forms the stable ferric–superoxide–TyrB10/GlnE7 complex. This complex reacts with internalized NO with a bimolecular rate constant of 1010 M−1 s−1 forming nitrate, which migrates to the CD loop and unfurls the spring-like structure. To restart the cycle, LeuE11 toggles back to the ferric iron. Actuating electron transfer with O2 and NO movements averts irreversible NO poisoning and reductive inactivation of the enzyme. Together, structure snapshots and kinetic constants provide glimpses of intermediate conformational states, time scales for motion, and associated energies.

Displaying epitope B and epitope 7 of porcine reproductive and respiratory syndrome virus on virus like particles of porcine circovirus type 2 provides partial protection to pigs.

The Cap of porcine circovirus type 2 (PCV2) can be assembled into virus like particles (VLPs) in vitro that have multiple loops located on the particle surface. This would make it a good vehicle for displaying exogenous proteins or epitopes. We derived two epitopes, epitope B (EpB, S37HIQLIYNL45) and epitope 7 (Ep7, Q196WGRL200) from Gp5 of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). We replaced the core region of Loop CD (L75PPGGGSN82) and the carboxyl terminus (K222DPPL226) of PCV2 Cap, respectively, to construct a bi-epitope chimeric PCV2 Cap. Its immunogenicity and protective effects were evaluated as one PRRSV subunit vaccine. The chimeric PCV2 Cap was soluble, efficiently expressed in an Escherichia coli expression system, and could be self-assembled into chimeric virus like particles (cVLPs) with a diameter of 12-15 nm. Western blotting confirmed that the cVLPs could be specifically recognized by anti-PCV2, anti-EpB and anti-Ep7 antibodies. The cVLPs vaccine could alleviate the clinical symptoms and reduce the viral loads after HP-PRRSV challenge in 100-120 days old pigs. These data suggest that the cVLPs vaccine could provide pigs with partial protection against homologous PRRSV strains, and it provides a new design for additional PRRSV subunit vaccines.