Fractionation of sulfated galactan from the red alga Botryocladia occidentalis separates its anticoagulant and anti-SARS-CoV-2 properties

Sulfation pattern and molecular weight (MW) play a key role in the biological actions of sulfated glycans. Besides anticoagulant effects, certain sulfated glycans can also exhibit anti-SARS-CoV-2 properties. To develop a more selective antiviral carbohydrate, an efficient strategy to separate these two actions is required. In this work, low MW fractions derived from the red alga Botryocladia occidentalis sulfated galactan (BoSG) were generated, structurally characterized, and tested for activity against SARS-CoV-2 and blood coagulation. The lowest MW fraction was found to be primarily composed of octasaccharides of monosulfated monosaccharides. Unlike heparin or native BoSG, we found that hydrolyzed BoSG products had weak anticoagulant activities as seen by aPTT and inhibitory assays using purified cofactors. In contrast, lower MW BoSG-derivatives retained anti-SARS-CoV-2 activity using SARS-CoV-2 spike (S)-protein pseudotyped lentivirus vector in HEK-293T-hACE2 cells monitored by GFP. Surface plasmon resonance confirmed that longer chains are necessary for BoSG to interact with coagulation cofactors but is not required for interactions with certain S-protein variants. We observed distinct affinities of BoSG derivatives for the S-proteins of different SARS-CoV-2 strains, including WT, N501Y (Alpha), K417T/E484K/N501Y (Gamma), and L542R (Delta) mutants, and stronger affinity for the N501Y-containing variants. Docking of the four possible monosulfated BoSG disaccharides in interactions with the N501Y mutant S-protein predicted potential binding poses of the BoSG constructs and favorable binding in close proximity to the 501Y residue. Our results demonstrate that depolymerization and fractionation of BoSG are an effective strategy to segregate its anticoagulant property from its anti-SARS-CoV-2 action.     

Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling

The nonapeptide Leuprorelin, one of the LHRH agonists, was studied by means of 2D nuclear magnetic resonance spectroscopy and molecular modeling. NOESY spectra in aqueous/deuterated methanol solution (50%H₂O/CD₃OD) at low temperature (268 K) revealed short-range nOe connectivities (i, i+1), characteristic of flexibility of the molecule. The H^N–H^N sequential connectivities observed provide evidence that the sequence has the propensity to form a bend involving residues 5 and 6 and the N-terminal segment. The -proton chemical shifts compared to random coil and additional data from the amide proton temperature coefficients support this assumption. One long-range nOe cross peak between H₂ –H^NEth is indicative of proximity between C- and N-termini.     

Conformational analysis of the nonapeptide leuprorelin using NMR and molecular modeling

Summary The nonapeptide Leuprorelin, one of the LHRH agonists, was studied by means of 2D nuclear magnetic resonance spectroscopy and molecular modeling. NOESY spectra in aqueous/deuterated methanol solution (50% H₂O/CD₃OD) at low temperature (268 K) revealed short-range nOe connectivities (i, i+1), characteristic of flexibility of the molecule. The H ^N -H ^N sequential connectivities observed provide evidence that the sequence has the propensity to form a bend involving residues 5 and 6 and the N-terminal segment. The α-proton chemical shifts compared to random coil and additional data from the amide proton temperature coefficients support this assumption. One long-range nOe cross peak between H ₂ ^α -H ^NEth is indicative of proximity between C- and N-termini.     

A surface plasmon resonance assay for measurement of neuraminidase inhibition,sensitivity of wild‐type influenza neuraminidase and its H274Y mutant to the antiviral drugs zanamivir and oseltamivir

Antiviral resistance is currently monitored by a labelled enzymatic assay, which can give inconsistent results because of the short half‐life of the labelled product, and variations in assay conditions. In this paper, we describe a competitive surface plasmon resonance (SPR) inhibition assay for measuring the sensitivities of wild‐type neuraminidase (WT NA) and the H274Y (histidine 274 tyrosine) NA mutant to antiviral drugs. The two NA isoforms were expressed in High‐five™ (Trichoplusia ni) insect cells. A spacer molecule (1,6‐hexanediamine (HDA)) was conjugated to the 7‐hydroxyl group of zanamivir, and the construct (HDA‐zanamivir) was immobilized onto a SPR sensor chip to obtain a final immobilization response of 431 response units. The immobilized HDA‐zanamivir comprised a bio‐specific ligand for the WT and mutant proteins. The effects of the natural substrate (sialic acid) and two inhibitors (zanamivir and oseltamivir) on NA binding to the immobilized ligand were studied. The processed SPR data was analysed to determine 50% inhibitory concentrations (IC_50‐spr), using a log dose–response curve fit. Although both NA isoforms had almost identical IC_50‐spr values for sialic acid (WT = 5.5 nM; H274Y mutant = 3.25 nM) and zanamivir (WT = 2.16 nM; H274Y mutant = 2.42 nM), there were significant differences between the IC_50‐spr values obtained for the WT (7.7 nM) and H274Y mutant (256 nM) NA in the presence of oseltamivir, indicating that oseltamivir has a reduced affinity for the H274Y mutant. The SPR inhibition assay strategy presented in this work could be applied for the rapid screening of newly emerging variants of NA for their sensitivity to antiviral drugs. Copyright © 2015 John Wiley & Sons, Ltd.     

Performance,carcass traits and serum metabolomic profile of Nellore males with different genetic potential for post-weaning growth

The BW has been largely used as a selection criterion in genetic selection programmes; however, increases in BW can affect animal metabolism and metabolites. The knowledge of how genetic potential for growth affects the metabolites can give a footprint of growth metabolism. This research aimed to evaluate the effect of genetic potential for post-weaning growth (GG) on performance, carcass traits and serum metabolome of non-castrated Nellore males during the finishing phase. Forty-eight Nellore non-castrated males, with divergent potential for post-weaning growth, were selected and divided into two groups: high potential for post-weaning growth (HG; n = 24) and low potential for post-weaning growth (LG; n = 24). Animals were kept and fed for 90 days where performance and ultrasound carcass traits were evaluated. Blood samples were collected at the beginning and end of feeding period to analyse serum metabolites concentration. The hot carcass weight and dressing percentage were recorded at slaughter. The feedlot performance and carcass traits were not affected by genetic potential. The HG animals had a lower glucose (P = 0.039), glutamate (P = 0.038), glutamine (P = 0.004), greater betaine (P = 0.039) and pyruvate (P = 0.039) compared to the LG group at the beginning of feedlot. In addition, higher creatine phosphate concentrations were observed at the beginning of feeding period, compared to final, for both groups (P = 0.039). In conclusion, the genetic potential for post-weaning growth does not affect performance and carcass traits during the finishing period. Differences in metabolite concentrations can be better found at the beginning of feedlot, providing a footprint of growth metabolism, but similar metabolite concentration at the end of finishing period.     

Structural basis for the negative allostery between Ca(2+)- and Mg(2+)-binding in the intracellular Ca(2+)-receptor calbindin D9k.

The three-dimensional structures of the magnesium- and manganese-bound forms of calbindin D9k were determined to 1.6 A and 1.9 A resolution, respectively, using X-ray crystallography. These two structures are nearly identical but deviate significantly from both the calcium bound form and the metal ion-free (apo) form. The largest structural differences are seen in the C-terminal EF-hand, and involve changes in both metal ion coordination and helix packing. The N-terminal calcium binding site is not occupied by any metal ion in the magnesium and manganese structures, and shows little structural deviation from the apo and calcium bound forms. 1H-NMR and UV spectroscopic studies at physiological ion concentrations show that the C-terminal site of the protein is significantly populated by magnesium at resting cell calcium levels, and that there is a negative allosteric interaction between magnesium and calcium binding. Calcium binding was found to occur with positive cooperativity at physiological magnesium concentration.     

Dynamic identification of H2 epitopes from Leishmania (Leishmania) amazonensis cysteine proteinase B with potential immune activity during murine infection

Peptides from the COOH‐terminal extension of cysteine proteinase B from Leishmania (Leishmania) amazonensis (cyspep) can modulate immune responses in vertebrate hosts. With this hypothesis as base, we used the online analysis tool SYFPEITHI to predict seven epitopes from this region with potential to bind H2 proteins. We performed proliferation tests and quantified reactive T lymphocytes applying a cytometry analysis, using samples from draining lymph node of lesions from L. (L.) amazonensis‐infected mice. To define reactivity of T cells, we used complexes of DimerX (H2 D^b:Ig and H2 L^d:Ig) and the putative epitopes. Additionally, we applied surface plasmon resonance to verify real time interactions between the putative epitopes and DimerX proteins. Five peptides induced blastogenesis in BALB/c cells, while only two presented the same property in C57BL/6 mouse cells. In addition, our data indicate the existence of CD8⁺ T lymphocyte populations able to recognize each tested peptide in both murine strains. We observed an overlapping of results between the peptides that induced lymphocyte proliferation and those capable of binding to the DimerX in the surface plasmon resonance assays thus indicating that using these recombinant proteins in biosensing analyses is a promising tool to study real time molecular interactions in the context of major histocompatibility complex epitopes. The data gathered in this study reinforce the hypothesis that cyspep‐derived peptides are important factors in the murine host infection by L. (L.) amazonensis. Copyright © 2014 John Wiley & Sons, Ltd.     

Convergent evolution of a parasite-encoded complement control protein-scaffold to mimic binding of mammalian TGF-β to its receptors,TβRI and TβRII

The mouse intestinal helminth Heligmosomoides polygyrus modulates host immune responses by secreting a transforming growth factor (TGF)-β mimic (TGM), to expand the population of Foxp3⁺ T_regs. TGM comprises five complement control protein (CCP)-like domains, designated D1-D5. Though lacking homology to TGF-β, TGM binds directly to the TGF-β receptors TβRI and TβRII and stimulates the differentiation of naïve T-cells into T_regs. However, the molecular determinants of binding are unclear. Here, we used surface plasmon resonance, isothermal calorimetry, NMR spectroscopy, and mutagenesis to investigate how TGM binds the TGF-β receptors. We demonstrate that binding is modular, with D1-D2 binding to TβRI and D3 binding to TβRII. D1-D2 and D3 were further shown to compete with TGF-β(TβRII)₂ and TGF-β for binding to TβRI and TβRII, respectively. The solution structure of TGM-D3 revealed that TGM adopts a CCP-like fold but is also modified to allow the C-terminal strand to diverge, leading to an expansion of the domain and opening potential interaction surfaces. TGM-D3 also incorporates a long structurally ordered hypervariable loop, adding further potential interaction sites. Through NMR shift perturbations and binding studies of TGM-D3 and TβRII variants, TGM-D3 was shown to occupy the same site of TβRII as bound by TGF-β using both a novel interaction surface and the hypervariable loop. These results, together with the identification of other secreted CCP-like proteins with immunomodulatory activity in H. polygyrus, suggest that TGM is part of a larger family of evolutionarily plastic parasite effector molecules that mediate novel interactions with their host.     

A discrete domain of the human TrkB receptor defines the binding sites for BDNF and NT-4

TrkB is a member of the Trk family of tyrosine kinase receptors. In vivo, the extracellular region of TrkB is known to bind, with high affinity, the neurotrophin protein brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4). We describe the expression and purification of the second Ig-like domain of human TrkB (TrkBIg(2)) and show, using surface plasmon resonance, that this domain is sufficient to bind BDNF and NT-4 with subnanomolar affinity. BDNF and NT-4 may have therapeutic implications for a variety of neurodegenerative diseases. The specificity of binding of the neurotrophins to their receptor TrkB is therefore of interest. We examine the specificity of TrkBIg(2) for all the neurotrophins, and use our molecular model of the BDNF-TrkBIg(2) complex to examine the residues involved in binding. It is hoped that the understanding of specific interactions will allow design of small molecule neurotrophin mimetics.     

Ligand-binding regulation of LXR/RXR and LXR/PPAR heterodimerizations: SPR technology-based kinetic analysis correlated with molecular dynamics simulation

Liver X receptor (LXR) and peroxisome proliferator-activated receptor (PPAR) are two members of nuclear receptors involved in the nutrient metabolisms of dietary fatty acid and cholesterol. They are found to be of cross-talk function in that LXR regulates fatty acid synthesis and PPAR controls fatty acid degradation. LXRs (LXRalpha and LXRbeta) function by forming obligate heterodimers with the retinoid X receptor (RXR), and subsequently binding to specific DNA response elements within the regulatory regions of their target genes. In this work, the kinetic features concerning LXR/RXR and LXR/PPAR interactions have been fully investigated using surface plasmon resonance (SPR) technology. It is found that LXRs could bind to all the three PPAR subtypes, PPARalpha, PPARgamma and PPARdelta with different binding affinities, and such receptor/receptor interactions could be regulated by ligand binding. Moreover, molecular dynamics (MD) simulations were performed on six typical complex models. The results revealed that ligands may increase the interaction energies between the receptor interfaces of the simulated receptor/receptor complexes. The MD results are in agreement with the SPR data. Further analyses on the MD results indicated that the ligand binding might increase the hydrogen bonds between the interfaces of the receptor/receptor complex.     

Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors

Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional “three-finger” snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by “three-finger” snake neurotoxins.     

SPR and NMR characterization of the molecular interaction between A9 peptide and a model system of HER2 receptor: A fragment approach for selecting peptide structures specific for their target

The binding process of A9 peptide toward HER2‐DIVMP, a synthetic model of the receptor domain IV, was studied by using the surface plasmon resonance (SPR) technique, with the aim of validating it as a fast and reliable screening method for selecting peptide ligands specifically targeting a domain of their target. To investigate the structural basis of A9 binding to the model of HER2‐DIVMP, multiple ligand‐based nuclear magnetic resonance (NMR) methods were applied. The use of saturation transfer difference (STD) and WaterLOGSY NMR experiments identified key residues in the peptide for the receptor binding. Moreover, the bound conformation of the A9 peptide was obtained using transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments. The NMR data revealed an extended binding surface that confirms an in silico model previously reported. These structural findings could provide good starting points for future lead structures optimization specific for the receptor target.     

Structural and functional characterization of CFE88: evidence that a conserved and essential bacterial protein is a methyltransferase

CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227-residue protein has minimal sequence similarity to proteins of known 3D structure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase. Characterization of the conformation and function of CFE88 has been performed by using several techniques. Backbone atom and limited side-chain atom NMR resonance assignments have been obtained. The data indicate that CFE88 has two domains: an N-terminal domain with 163 residues and a C-terminal domain with 64 residues. The C-terminal domain is primarily helical, while the N-terminal domain has a mixed helical/extended (Rossmann) fold. By aligning the experimentally observed elements of secondary structure, an initial unrefined model of CFE88 has been constructed based on the X-ray structure of ErmC' methyltransferase (Protein Data Bank entry 1QAN). NMR and biophysical studies demonstrate binding of S-adenosyl-L-homocysteine (SAH) to CFE88; these interactions have been localized by NMR to the predicted active site in the N-terminal domain. Mutants that target this predicted active site (H26W, E46R, and E46W) have been constructed and characterized. Overall, our results both indicate that CFE88 is a methyltransferase and further suggest that the methyltransferase activity is essential for bacterial survival.     

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro³¹⁴–Trp³¹⁶ turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro³¹⁴ of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function.     

Empirical rules for rationalising visible circular dichroism of Cu2+ and Ni2+ histidine complexes: applications to the prion protein

A natively unfolded region of the prion protein, PrP(90-126) binds Cu(2+) ions and is vital for prion propagation. Pentapeptides, acyl-GGGTH(92-96) and acyl-TNMKH(107-111), represent the minimum motif for this Cu(2+) binding region. EPR and (1)H NMR suggests that the coordination geometry for the two binding sites is very similar. However, the visible CD spectra of the two sites are very different, producing almost mirror image spectra. We have used a series of analogues of the pentapeptides containing His(96) and His(111) to rationalise these differences in the visible CD spectra. Using simple histidine-containing tri-peptides we have formulated a set of empirical rules that can predict the appearance of Cu(2+) visible CD spectra involving histidine and amide main-chain coordination.     

Molecular modeling and dynamic simulations of agglutinin-like family members from Candida albicans: New insights into potential targets for the treatment of candidiasis

Infections by Candida albicans in immune compromised patients cause significant morbidity and mortality. In the search for potential molecular targets for drug development, the family of agglutinin-like proteins (Als) in C. albicans have been identified due to numerous attributes associated with high virulence, most prominently due to their role in adherence. Here, molecular models of individual members of the Als family illustrated common and unique structure features. Additionally, dynamic simulations were performed to display regions of high mobility. The results showed variations between Als members in the fluctuation of the A1B1 protein loop, which is located at the entrance to the peptide binding cavity, suggesting that this feature may be a factor contributing to observed differences in affinities to ligands and adhesion properties. Molecular docking results further suggested that ligand affinity could be influenced by movements in the A1B1 loop. In addition, a new site was identified in Als in an area adjacent to the peptide binding cavity that could serve as a new binding site for the design of future anti-adhesion ligands that provide increased specificity inhibiting Als proteins from C. albicans.     

The C-terminal and beta-wing regions of ammodytoxin A, a neurotoxic phospholipase A2 from Vipera ammodytes ammodytes, are critical for binding to factor Xa and for anticoagulant effect

Ammodytoxin A (AtxA) from the venom of Vipera ammodytes ammodytes belongs to group IIA secreted phospholipase A2 (sPLA2), for which the major pathologic activity is presynaptic neurotoxicity. We show here that this toxin also affects hemostasis because it exhibits strong anticoagulant activity. AtxA binds directly to human coagulation factor Xa (FXa) with Kdapp of 32 nM, thus inhibiting the activity of the prothrombinase complex with an IC50 of 20 nM. To map the FXa-interaction site on AtxA, various mutants of AtxA produced by site-directed mutagenesis and expressed in Escherichia coli were tested in the study. In surface plasmon resonance (SPR) measurements, with FXa covalently attached to the sensor chip, we show that the FXa-binding site on AtxA includes several basic amino acid residues at the C-terminal and beta-wing regions of the molecule. Applying an in vitro biological test for inhibition of prothrombinase activity, we further demonstrate that the same residues are also very important for the anticoagulant activity of AtxA. We conclude that the anticoagulant site of AtxA is located in the C-terminal and beta-wing regions of this phospholipase A2. Synthetic peptides comprising residues of the deduced anticoagulant site of AtxA provide a basis to synthesize novel anticoagulant drugs.