Identification of Sperm-Binding Sites in the N-Terminal Domain of Bovine Egg Coat Glycoprotein ZP4

The species-selective interaction between sperm and egg at the beginning of mammalian fertilisation is partly mediated by a transparent envelope called the zona pellucida (ZP). The ZP is composed of three or four glycoproteins (ZP1–ZP4). The functions of the three proteins present in mice (ZP1–ZP3) have been extensively studied. However, the biological role of ZP4, which was found in all other mammals studied so far, has remained largely unknown. Previously, by developing a solid support assay system, we showed that ZP4 exhibits sperm-binding activity in bovines and the N-terminal domain of bovine ZP4 (bZP4 ZP-N1 domain) is a sperm-binding region. Here, we show that bovine sperm bind to the bZP4 ZP-N1 domain in a species-selective manner and that N-glycosylation is not required for sperm-binding activity. Moreover, we identified three sites involved in sperm binding (site I: from Gln-41 to Pro-46, site II: from Leu-65 to Ser-68 and site III: from Thr-108 to Ile-123) in the bZP4 ZP-N1 domain using chimeric bovine/porcine and bovine/human ZP4 recombinant proteins. These results provide in vitro experimental evidence for the role of the bZP4 ZP-N1 domain in mediating sperm binding to the ZP.

Monomodular Pseudomonas aeruginosa phage JG004 lysozyme (Pae87) contains a bacterial surface-active antimicrobial peptide-like region and a possible substrate-binding subdomain

Phage lysins are a source of novel antimicrobials to tackle the bacterial antibiotic resistance crisis. The engineering of phage lysins is being explored as a game-changing technological strategy for introducing a more precise approach in the way we apply antimicrobial therapy. Such engineering efforts will benefit from a better understanding of lysin structure and function. In this work, the antimicrobial activity of the endolysin from Pseudomonas aeruginosa phage JG004, termed Pae87, has been characterized. This lysin had been previously identified as an antimicrobial agent candidate, able to interact with the Gram-negative surface and disrupt it. Further evidence is hereby provided on this matter, based on a structural and biochemical study. A high-resolution crystal structure of Pae87 complexed with a peptidoglycan fragment showed a separate substrate-binding region within the catalytic domain, 18 Å away from the catalytic site and located at the opposite side of the lysin molecule. This substrate binding region was conserved among phylogenetically related lysins lacking an additional cell wall binding domain, but not among those containing such a module. Two glutamic acids were identified as relevant for the peptidoglycan degradation activity, although Pae87 antimicrobial activity was seemingly unrelated to it. In contrast, an antimicrobial peptide-like region within Pae87 C-terminus, named P87, was found to be able to actively disturb the outer membrane and have antibacterial activity by itself. Therefore, we propose an antimicrobial mechanism for Pae87 in which the P87 peptide plays the role of binding to the outer membrane and disrupting the cell wall function, either with or without the participation of Pae87 catalytic activity.

NOG-Derived Peptides Can Restore Neuritogenesis on a CRASH Syndrome Cell Model

Homo- and heterophilic binding mediated by the immunoglobulin (Ig)-like repeats of cell adhesion molecules play a pivotal role in cell-cell and cell-extracellular matrix interactions. L1CAM is crucial to neuronal differentiation, in both mature and developing nervous systems, and several studies suggest that its functional interactions are mainly mediated by Ig2–Ig2 binding. X-linked mutations in the human L1CAM gene are summarized as L1 diseases, including the most diagnosed CRASH neurodevelopmental syndrome. In silico simulations provided a molecular rationale for CRASH phenotypes resulting from mutations I179S and R184Q in the homophilic binding region of Ig2. A synthetic peptide reproducing such region could both mimic the neuritogenic capacity of L1CAM and rescue neuritogenesis in a cellular model of the CRASH syndrome, where the full L1CAM ectodomain proved ineffective. Presented functional evidence opens the route to the use of L1CAM-derived peptides as biotechnological and therapeutic tools.

Structural and functional characterizations of altered infectivity and immune evasion of SARS-CoV-2 Omicron variant

The SARS-CoV-2 Omicron with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the Spike (S) from Omicron reveals amino acid substitutions forging new interactions that stably maintain an active conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of viral fusion step. Alterations in local conformation, charge and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Apart from already existing mutations, we have identified three new immune escape sites: 1) Q493R, 2) G446S and 3) S371L/S373P/S375F that confers greater resistance to five of the six classes of RBD-antibodies. Structure of the Omicron S bound with human ACE2, together with analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.

H3N2 Influenza Viruses with 12- or 16-Amino Acid Deletions in the Receptor-Binding Region of Their Hemagglutinin Protein

The hemagglutinin (HA) protein of influenza viruses serves as the receptor-binding protein and is the principal target of the host immune system. The antigenic epitopes in the receptor-binding region are known to tolerate mutations, but here, we show that even deletions of 12 or 16 amino acids in this region can be accommodated.

Force-induced changes of α-catenin conformation stabilize vascular junctions independent of vinculin

Cadherin-mediated cell adhesion requires anchoring via the β-catenin-α-catenin complex to the actin cytoskeleton, yet, α-catenin binds F-actin only weakly. A covalent fusion of VE-cadherin to α-catenin enhances actin anchorage in endothelial cells and strongly stabilizes endothelial junctions in vivo, blocking inflammatory responses. Here, we have analyzed the underlying mechanism. We found that VE-cadherin-α-catenin constitutively recruits the actin adaptor vinculin. However, removal of the vinculin binding region of α-catenin did not impair the ability of VE-cadherin-α-catenin to enhance junction integrity. Searching for an alternative explanation for the junction stabilizing mechanism, we found that an antibody-defined epitope, normally buried in a short α1-helix of the actin binding domain (ABD) of α-catenin, is openly displayed in junctional VE-cadherin-α-catenin chimera. This epitope, we found to become exposed in normal α-catenin upon triggering thrombin-induced tension across the VE-cadherin complex. These results suggest, that the VE-cadherin-α-catenin chimera stabilizes endothelial junctions due to conformational changes in the ABD of α-catenin, which support constitutive strong binding to actin.

Substrate recognition determinants of human eIF2α phosphatases

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Here, we generated a series of R15 truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A 325–554 and R15B 340–639 . G-actin, which has been proposed to confer selectivity to R15 phosphatases, does not bind these regions, indicating that it is not required for substrate binding. Fragments containing the substrate-binding regions but lacking the PP1-binding motif trapped the phospho-substrate and caused accumulation of phosphorylated eIF2α in unstressed cells. Activity assays in cells showed that R15A 325–674 and R15B 340–713 , encompassing the substrate-binding region and the PP1-binding region, exhibit wild-type activity. This work identifies the substrate-binding region in R15s, that functions as a phospho-substrate trapping mutant, thereby defining a key region of R15s for follow up studies.

Cryo-EM structure of the octameric pore of Clostridium perfringens β-toxin

We describe the cryo-EM structure of Clostridium perfringens β-toxin (CBP) in styrene maleic acid (SMA) discs, which represents the membrane-inserted pore form, at near atomic resolution. We show that CPB forms an octamer, which though having a similar conformation to the hetero-oligomeric pores of bicomponent leukocidins, features a different receptor binding region and a novel N-terminal β-barrel. The latter contains an additional selectivity filter and creates a bipolar pore. We propose that the N-terminal β-barrel domain may regulate oligomerization and solubility of the complex and influence channel conductance and monomer stability. In addition, we show that the β-barrel protrusion domain can be modified or exchanged without affecting the pore forming ability, thus making the pore particularly attractive for macromolecule sensing and nanotechnology. The cryo-EM structure of the octameric pore of CPB will facilitate future developments in both nanotechnology and basic research.

Protection of mice against experimental cryptococcosis by synthesized peptides delivered in glucan particles

The high global burden of cryptococcosis has made development of a protective vaccine a public health priority. We previously demonstrated that a vaccine composed of recombinant Cryptococcus neoformans chitin deacetylase 2 (Cda2) delivered in glucan particles (GPs) protects BALB/c and C57BL/6 mice from an otherwise lethal challenge with a highly virulent C. neoformans strain. An immunoinformatic analysis of Cda2 revealed a peptide sequence predicted to have strong binding to the MHC Class II (MHC II) H2-IAd allele found in BALB/c mice. BALB/c mice vaccinated with GPs containing a 32 amino acid peptide (Cda2-Pep1) that included this strong binding region were protected from cryptococcosis. Protection was lost with GP-based vaccines containing versions of recombinant Cda2 protein and Cda2-Pep1 with mutations predicted to greatly diminish MHC II binding. Cda2 has homology to the three other C. neoformans chitin deacetylases, Cda1, Cda3 and Fpd1, in the high MHC II binding region. GPs loaded with homologous peptides of Cda1, Cda3 and Fpd1 protected BALB/c mice from experimental cryptococcosis, albeit not as robustly as the Cda2-Pep1 vaccine. Finally, seven other peptides were synthesized based on regions in Cda2 predicted to contain promising CD4+ T cell epitopes in BALB/c or C57BL/6 mice. While five peptide vaccines significantly protected BALB/c mice, only one protected C57BL/6 mice. Thus, GP-based vaccines containing a single peptide can protect mice against cryptococcosis. However, given the diversity of human MHC II alleles, a peptide-based Cryptococcus vaccine for use in humans would be challenging and likely need to contain multiple peptide sequences.

Genetic Diversity of Plasmodium vivax Reticulocyte Binding Protein 2b in Global Parasite Populations

BackgroundPlasmodium vivax reticulocyte binding protein 2b (PvRBP2b) plays a critical role in parasite invasion of reticulocytes by binding the transferring receptor 1. PvRBP2b is a vaccine candidate since the antibody titers against PvRBP2b recombinant proteins are negatively correlated with the parasitemia and risk of vivax malaria. This study aims to analyze the genetic diversity of the PvRBP2b gene in the global P. vivax populations. MethodsThe near full-length PvRBP2b nucleotide sequences (190-8349 bp) were obtained from 88 P. vivax isolates collected from the China–Myanmar border (n=44) and Thailand (n=44). Additional 224 sequences of PvRBP2b were retrieved from genome sequences from the global parasite populations. The genetic diversity, neutral selections, haplotypes distribution and genetic differentiation of PvRBP2b were examined. ResultsThe genetic diversity of PvRBP2b was distributed unevenly with the peak in the reticulocyte binding region in the N-terminus and subjected to the balancing selection. Several amino acid variants were found in all or nearly all endemic fields. However, the critical residues responsible for reticulocyte binding were highly conserved. There was substantial population differentiation according to the geographical separation. The distribution of haplotypes in the reticulocyte binding region varied among regions; even the two major haplotypes Hap_6 and Hap_8 were found in only five populations. ConclusionsOur data showed considerable genetic variations of PvRBPb in global parasite populations, and the geographic divergence may pose a challenge to PvRBP2b-based vaccine development.