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Researchers from the world over are working to create prophylactic and therapeutic interventions to combat the COVID-19 global healthcare crisis. The current therapeutic options against the COVID-19 include repurposed drugs aimed at targets other than virus-specific proteins. Antibody-based therapeutics carry a lot of promise, and there are several of these candidates for COVID-19 treatment currently being investigated in the preclinical and clinical research stages around the world. The viral spike protein (S protein) appears to be the main target of antibody development candidates, with the majority being monoclonal antibodies. Several antibody candidates targeting the SARS-CoV-2 S protein include LY-CoV555, REGN-COV2, JS016, TY027, CT-P59, BRII-196, BRII-198 and SCTA01. These neutralizing antibodies will treat COVID-19 and possibly future coronavirus infections. Future studies should focus on effective immune-therapeutics and immunomodulators with the purpose of developing specific, affordable, and cost-effective prophylactic and treatment regimens to fight the COVID-19 globally.

Prion diseases in mammals are caused by a conformational transition of the cellular prion protein from its native conformation (PrP(C)) to a pathological isoform called “prion protein scrapie” (PrP(Sc)). A molecular level of understanding of this conformational transition will be helpful in unveiling the disease etiology. Experimental structural biological techniques (NMR and X‐ray crystallography) have been used to unravel the atomic level structural information for the prion and its binding partners. More than one hundred three‐dimensional structures of the mammalian prions have been deposited in the protein databank. Structural studies on the prion protein and its structural transitions will deepen our understanding of the molecular basis of prion pathogenesis and will provide valuable guidance for future structure‐based drug discovery endeavors.

Conversion of the cellular prion protein PrPC into its pathogenic isoform PrPSc is the hallmark of prion diseases, fatal neurodegenerative diseases affecting many mammalian species including humans. Anti‐prion monoclonal antibodies can arrest the progression of prion diseases by stabilizing the cellular form of the prion protein. Here, we present the crystal structure of the POM6 Fab fragment, in complex with the mouse prion protein (moPrP). The prion epitope of POM6 is in close proximity to the epitope recognized by the purportedly toxic antibody fragment, POM1 Fab also complexed with moPrP. The POM6 Fab recognizes a larger binding interface indicating a likely stronger binding compared to POM1. POM6 and POM1 exhibit distinct biological responses. Structural comparisons of the bound mouse prion proteins from the POM6 Fab:moPrP and POM1 Fab:moPrP complexes reveal several key regions of the prion protein that might be involved in initiating mis‐folding events.

Prion diseases are progressive, infectious neurodegenerative disorders caused primarily by the misfolding of the cellular prion protein (PrPc) into an insoluble, protease-resistant, aggregated isoform termed PrPsc. In native conditions, PrPc has a structured C-terminal domain and a highly flexible N-terminal domain. A part of this N-terminal domain consists of 4–5 repeats of an unusual glycine-rich, eight amino acids long peptide known as the octapeptide repeat (OR) domain. In this article, we successfully report the first crystal structure of an OR of PrPc bound to the Fab fragment of the POM2 antibody. The structure was solved at a resolution of 2.3 A by molecular replacement. Although several studies have previously predicted a β-turn-like structure of the unbound ORs, our structure shows an extended conformation of the OR when bound to a molecule of the POM2 Fab indicating that the bound Fab disrupts any putative native β turn conformation of the ORs. Encouraging results from several recent studies have shown that administering small molecule ligands or antibodies targeting the OR domain of PrP result in arresting the progress of peripheral prion infections both in ex vivo and in in vivo models. This makes the structural study of the interactions of POM2 Fab with the OR domain very important as it would help us to design smaller and tighter binding OR ligands.

Pea abscisic acid responsive (ABR17) protein is a member of the pathogenesis-related 10 (PR10) family of proteins and its ribonuclease (RNase) activity has been reported previously. In order to investigate the amino acids important for the demonstrated ribonuclease activity of ABR17, site-directed mutants H69L and E148A were generated, expressed in Escherichia coli and purified to homogeneity. These mutations affected RNase activity differently; the H69L mutant exhibited a decreased RNase activity whereas E148A exhibited an elevated activity. A structural model for pea ABR17 has been generated using the three dimensional structure of Lupinus luteus PR10 protein in order to explain the possible effects of the H69L and the E148A mutations on substrate binding and catalysis.

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) plays an essential role in nitroglycerin (GTN) bioactivation, resulting in formation of NO or a related activator of soluble guanylate cyclase. ALDH2 denitrates GTN to 1,2-glyceryl dinitrate and nitrite but also catalyzes reduction of GTN to NO. To elucidate the relationship between ALDH2-catalyzed GTN bioconversion and established ALDH2 activities (dehydrogenase, esterase), we compared the function of the wild type (WT) enzyme with mutants lacking either the reactive Cys-302 (C302S) or the general base Glu-268 (E268Q). Although the C302S mutation led to90% loss of all enzyme activities, the E268Q mutant exhibited virtually unaffected rates of GTN denitration despite low dehydrogenase and esterase activities. The nucleotide co-factor NAD caused a pronounced increase in the rates of 1,2-glyceryl dinitrate formation by WT-ALDH2 but inhibited the reaction catalyzed by the E268Q mutant. GTN bioactivation measured as activation of purified soluble guanylate cyclase or release of NO in the presence of WT- or E268Q-ALDH2 was markedly potentiated by superoxide dismutase, suggesting that bioavailability of GTN-derived NO is limited by co-generation of superoxide. Formation of superoxide was confirmed by determination of hydroethidine oxidation that was inhibited by superoxide dismutase and the ALDH2 inhibitor chloral hydrate. E268Q-ALDH2 exhibited approximately 50% lower rates of superoxide formation than the WT enzyme. Our results suggest that Glu-268 is involved in the structural organization of the NAD-binding pocket but is not required for GTN denitration. ALDH2-catalyzed superoxide formation may essentially contribute to oxidative stress in GTN-exposed blood vessels.

Dipeptidyl-peptidases III (DPP III) are zinc-dependent enzymes that specifically cleave the first two amino acids from the N terminus of different length peptides. In mammals, DPP III is associated with important physiological functions and is a potential biomarker for certain types of cancer. Here, we present the 1.95-Å ; crystal structure of yeast DPP III representing the prototype for the M49 family of metallopeptidases. It shows a novel fold with two domains forming a wide cleft containing the catalytic metal ion. DPP III exhibits no overall similarity to other metallopeptidases, such as thermolysin and neprilysin, but zinc coordination and catalytically important residues are structurally conserved. Substrate recognition is accomplished by a binding site for the N terminus of the peptide at an appropriate distance from the metal center and by a series of conserved arginine residues anchoring the C termini of different length substrates.

Misfolded prion proteins are the cause of neurodegenerative diseases that affect many mammalian species, including humans. Transmission of the prion diseases poses a considerable public-health risk as a specific prion disease such as bovine spongiform encephalopathy can be transferred to humans and other mammalian species upon contaminant exposure. The underlying mechanism of prion propagation and the species barriers that control cross species transmission has been investigated quite extensively. So far a number of prion strains have been characterized and those have been intimately linked to species-specific infectivity and other pathophysiological manifestations. These strains are encoded by a protein-only agent, and have a high degree of sequence identity across mammalian species. The molecular events that lead to strain differentiation remain elusive. In order to contribute to the understanding of strain differentiation, we have determined the crystal structures of the globular, folded domains of four prion proteins (cow, deer, elk and Syrian hamster) bound to the POM1 antibody fragment Fab. Although the overall structural folds of the mammalian prion proteins remains extremely similar, there are several local structural variations observed in the misfolding-initiator motifs. In additional molecular dynamics simulation studies on these several prion proteins reveal differences in the local fluctuations and imply that these differences have possible roles in the unfolding of the globular domains. These local variations in the structured domains perpetuate diverse patterns of prion misfolding and possibly facilitate the strain selection and adaptation.

Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrP(c) to the pathogenic isoform PrP(sc). Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120-232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å, β = 95.6°.

Conformational transitions of the cellular form of the prion protein, PrP(C), into an infectious isoform, PrP(Sc), are considered to be central events in the progression of fatal neurodegenerative diseases known as transmissible spongiform encephalopathies. Tricyclic phenothiazine compounds exhibit antiprion activity; however, the underlying molecular mechanism of PrP(Sc) inhibition remains elusive. We report the molecular structures of two phenothiazine compounds, promazine and chlorpromazine bound to a binding pocket formed at the intersection of the structured and the unstructured domains of the mouse prion protein. Promazine binding induces structural rearrangement of the unstructured region proximal to β1, through the formation of a "hydrophobic anchor." We demonstrate that these molecules, promazine in particular, allosterically stabilize the misfolding initiator-motifs such as the C terminus of α2, the α2-α3 loop, as well as the polymorphic β2-α2 loop. Hence, the stabilization effects of the phenothiazine derivatives on initiator-motifs induce a PrP(C) isoform that potentially resists oligomerization.

Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.

Prion diseases are neurodegenerative diseases characterized by the conversion of the cellular prion protein PrPcinto a pathogenic isoform PrPsc. Passive immunization with antiprion monoclonal antibodies can arrest the progression of prion diseases. Here, the crystal structure of the Fab fragment of an antiprion monoclonal antibody, POM1, in complex with human prion protein (huPrPc) has been determined to 2.4 Å resolution. The prion epitope of POM1 is in close proximity to the epitope recognized by the purportedly therapeutic antibody fragment ICSM18 Fab in complex with huPrPc. POM1 Fab forms a 1:1 complex with huPrPcand the measuredKdof 4.5 × 10−7 Mreveals moderately strong binding between them. Structural comparisons have been made among three prion–antibody complexes: POM1 Fab–huPrPc, ICSM18 Fab–huPrPcand VRQ14 Fab–ovPrPc. The prion epitopes recognized by ICSM18 Fab and VRQ14 Fab are adjacent to a prion glycosylation site, indicating possible steric hindrance and/or an altered binding mode to the glycosylated prion proteinin vivo. However, both of the glycosylation sites on huPrPcare positioned away from the POM1 Fab binding epitope; thus, the binding mode observed in this crystal structure and the binding affinity measured for this antibody are most likely to be the same as those for the native prion proteinin vivo.

Saccharomyces cerevisiae squalene epoxidase contains two highly conserved motifs, 1 and 2, of unknown function. Amino acid substitutions in both regions reduce enzyme activity and/or alter allylamine sensitivity. In the homology model, these motifs flank the flavin adenine dinucleotide cofactor and form part of the interface between cofactor and substrate binding domains.

Squalene epoxidase (SE) is the target of terbinafine, which specifically inhibits the fungal enzyme in a noncompetitive manner. On the basis of functional homologies to p -hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens , the Erg1 protein contains two flavin adenine dinucleotide (FAD) domains and one nucleotide binding (NB) site. By in vitro mutagenesis of the ERG1 gene, which codes for the Saccharomyces cerevisiae SE, we isolated erg1 alleles that conferred increased terbinafine sensitivity or that showed a lethal phenotype when they were expressed in erg1 -knockout strain KLN1. All but one of the amino acid substitutions affected conserved FAD/nucleotide binding sites. The G 25 S, D 335 X (W, F, P), and G 210 A substitutions in the FADI, FADII, and NB sites, respectively, rendered the SE variants nonfunctional. The G 30 S and L 37 P variants exhibited decreased enzymatic activity, accompanied by a sevenfold increase in erg1 mRNA levels and an altered sterol composition, and rendered KLN1 more sensitive not only to allylamines (10 to 25 times) but also to other ergosterol biosynthesis inhibitors. The R 269 G variant exhibited moderately reduced SE activity and a 5- to 10-fold increase in allylamine sensitivity but no cross-sensitivity to the other ergosterol biosynthesis inhibitors. To further elucidate the roles of specific amino acids in SE function and inhibitor interaction, a homology model of Erg1p was built on the basis of the crystal structure of PHBH. All experimental data obtained with the sensitive Erg1 variants support this model. In addition, the amino acids responsible for terbinafine resistance, although they are distributed along the sequence of Erg1p, cluster on the surface of the Erg1p model, giving rise to a putative binding site for allylamines.

Prion diseases are fatal neurodegenerative diseases that affect humans and other animals. A conformational transition of the cellular prion protein, PrPC, into an infectious isoform, PrPSc, is the central event leading to aggregation and the fatal progression of these diseases. One of the therapeutic approaches for the prion diseases is the use of pharmacological chaperones. These molecules can stabilize the prion protein in its native conformation and can arrest the disease progression. Tricyclic phenothiazine compounds exhibit anti-prion activity; however, the underlying molecular mechanism of PrPSc inhibition remains elusive. We have determined the molecular structures of promazine and chlorpromazine bound to the mouse prion protein (moPrP) by forming crystals of the ternary complexes of the POM1 Fab:moPrP:promazine and the POM1 Fab:moPrP:chlorpromazine. The structures were solved by X-ray crystallography to resolutions of 1.9 Å and 2.2 Å, respectively. The small molecules are bound in a novel binding pocket formed at the intersection of the structured and the unstructured domains of the moPrP. Promazine binding induces a structural rearrangement of a portion of the unstructured region proximal to the first β-strand, β1, through the formation of a "hydrophobic anchor". We demonstrate that these molecules, promazine in particular, allosterically stabilize the misfolding initiator-motifs such as C-terminus of helix, α2, the α2-α3 loop as well as the polymorphic β2-α2 loop. Hence, the stabilization effects of the phenothiazine derivatives on initiator-motifs, induce a PrPC isoform that potentially resists oligomerization. Subtle structural differences are observed in the so-called initiator-motifs of the prion proteins that belong to many different mammalian species, and these diversities may possibly explain the generation of wide variety of scrapie strains in prion diseases.

Matrix metalloproteinase-2 (MMP-2) is a key intra- and extra-cellular protease which contributes to several oxidative stress related pathologies. A molecular understanding of 72 kDa MMP-2 activity, directly mediated by S-glutathiolation of its cysteine residues in the presence of peroxynitrite (ONOO(-)) and by phosphorylation of its serine and threonine residues, is essential to develop new generation inhibitors of intracellular MMP-2. Within its propeptide and collagen binding domains there is an interesting juxtaposition of predicted phosphorylation sites with nearby cysteine residues which form disulfide bonds. However, the combined effect of these two post-translational modifications on MMP-2 activity has not been studied. The activity of human recombinant 72 kDa MMP-2 (hrMMP-2) following in vitro treatments was measured by troponin I proteolysis assay and a kinetic activity assay using a fluorogenic peptide substrate. ONOO(-) treatment in the presence of 30 µM glutathione resulted in concentration-dependent changes in MMP-2 activity, with 0.1-1 µM increasing up to twofold and 100 µM attenuating its activity. Dephosphorylation of MMP-2 with alkaline phosphatase markedly increased its activity by sevenfold, either with or without ONOO(-). Dephosphorylation of MMP-2 also affected the conformational structure of the enzyme as revealed by circular dichroism studies, suggesting an increase in the proportion of α-helices and a decrease in β-strands compared to the phosphorylated form of MMP-2. These results suggest that ONOO(-) activation (at low µM) and inactivation (at high µM) of 72 kDa MMP-2, in the presence or absence of glutathione, is also influenced by its phosphorylation status. These insights into the role of post-translational modifications in the structure and activity of 72 kDa MMP-2 will aid in the development of inhibitors specifically targeting intracellular MMP-2.

Background Mitochondrial aldehyde dehydrogenase (ALDH2) plays an essential role in nitroglycerin (GTN) bioactivation, resulting in formation of nitric oxide (NO) or a related activator of soluble guanylate cyclase (sGC) and consequently in cGMP-mediated vasorelaxation [1]. ALDH2 denitrates GTN to 1,2-glyceryl dinitrate (1,2-GDN) and nitrite but also catalyzes reduction of GTN to nitric oxide (NO) [2]. To elucidate the mechanism of ALDH2-catalyzed GTN bioactivation in relation to the established ALDH2 activities (dehydrogenase, esterase), we compared the function of the wildtype (WT) enzyme with a mutant lacking the general base Glu268 (E268Q).