The fate of β-d-mannopyranose after its formation by endoplasmic reticulum α-(1→2)-mannosidase I catalysis

The automated docking program AutoDock was used to dock all 38 characteristic β-d-mannopyranose ring conformers into the active site of the yeast endoplasmic reticulum α-(1→2)-mannosidase I, a Family 47 glycoside hydrolase that converts Man9GlcNAc2 to Man8GlcNAc2. The subject of this work is to establish the conformational pathway that allows the cleaved glycon product to leave the enzyme active site and eventually reach the ground-state conformation. Twelve of the 38 conformers optimally dock in the active site where the inhibitors 1-deoxymannonojirimycin and kifunensine are found in enzyme crystal structures. A further 23 optimally dock in a second site on the side of the active-site well, while three dock outside the active-site cavity. It appears, through analysis of the internal energies of different ring conformations, of intermolecular energies between the ligands and enzyme, and of forces exerted on the ligands by the enzyme, that β-d-mannopyranose follows the path 3E→1C4→1H2→B2,5 before being expelled by the enzyme. The highly conserved second site that strongly binds β-d-mannopyranose-4C1 may exist to prevent competitive inhibition by the product, and is worthy of further investigation

Theory and computation show that Asp463 is the catalytic proton donor in human endoplasmic reticulum α-(1→2)-mannosidase I

It has been difficult to identify the proton donor and nucleophilic assistant/base of endoplasmic reticulum α-(1→2)-mannosidase I, a member of glycoside hydrolase Family 47, which cleaves the glycosidic bond between two α-(1→2)-linked mannosyl residues by the inverting mechanism, trimming Man9GlcNAc2 to Man8GlcNAc2 isomer B. Part of the difficulty is caused by the enzyme’s use of a water molecule to transmit the proton that attacks the glycosidic oxygen atom. We earlier used automated docking to conclusively determine that Glu435 in the yeast enzyme (Glu599 in the corresponding human enzyme) is the nucleophilic assistant. The commonly accepted proton donor has been Glu330 in the human enzyme (Glu132 in the yeast enzyme). However, for theoretical reasons this conclusion is untenable. Theory, automated docking of α-d-3S1-mannopyranosyl-(1→2)-α-d-4C1-mannopyranose and water molecules associated with candidate proton donors, and estimation of dissociation constants of the latter have shown that the true proton donor is Asp463 in the human enzyme (Asp275 in the yeast enzyme)

Selective engagement of FcγRIV by a M2e-specific single domain antibody construct protects against influenza A virus infection

Lower respiratory tract infections, such as infections caused by influenza A viruses, are a constant threat for public health. Antivirals are indispensable to control disease caused by epidemic as well as pandemic influenza A. We developed a novel anti-influenza A virus approach based on an engineered single-domain antibody (VHH) construct that can selectively recruit innate immune cells to the sites of virus replication. This protective construct comprises two VHHs. One VHH binds with nanomolar affinity to the conserved influenza A matrix protein 2 (M2) ectodomain (M2e). Co-crystal structure analysis revealed that the complementarity determining regions 2 and 3 of this VHH embrace M2e. The second selected VHH specifically binds to the mouse Fc gamma Receptor IV (Fc gamma RIV) and was genetically fused to the M2e-specific VHH, which resulted in a bi-specific VHH-based construct that could be efficiently expressed in Pichia pastoris. In the presence of M2 expressing or influenza A virus-infected target cells, this single domain antibody construct selectively activated the mouse Fc gamma RIV. Moreover, intranasal delivery of this bispecific Fc gamma RIV-engaging VHH construct protected wild type but not Fc gamma RIV-/- mice against challenge with an H3N2 influenza virus. These results provide proof of concept that VHHs directed against a surface exposed viral antigen can be readily armed with effector functions that trigger protective antiviral activity beyond direct virus neutralization

Developing methods for the production of antiviral VHH-Fc antibodies in Pichia pastoris for fast pandemic response

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The inhibitor endosidin 4 targets SEC7 domain-type ARF GTPase exchange factors and interferes with subcellular trafficking in eukaryotes

The trafficking of subcellular cargos in eukaryotic cells crucially depends on vesicle budding, a process mediated by ARFGEFs (ADP-ribosylation factor guanine nucleotide exchange factors). In plants, ARF-GEFs play essential roles in endocytosis, vacuolar trafficking, recycling, secretion, and polar trafficking. Moreover, they are important for plant development, mainly through controlling the polar subcellular localization of PIN-FORMED transporters of the plant hormone auxin. Here, using a chemical genetics screen in Arabidopsis thaliana, we identified Endosidin 4 (ES4), an inhibitor of eukaryotic ARF-GEFs. ES4 acts similarly to and synergistically with the established ARF-GEF inhibitor Brefeldin A and has broad effects on intracellular trafficking, including endocytosis, exocytosis, and vacuolar targeting. Additionally, Arabidopsis and yeast (Saccharomyces cerevisiae) mutants defective in ARF-GEF show altered sensitivity to ES4. ES4 interferes with the activation-based membrane association of the ARF1 GTPases, but not of their mutant variants that are activated independently of ARF-GEF activity. Biochemical approaches and docking simulations confirmed that ES4 specifically targets the SEC7 domain-containing ARF-GEFs. These observations collectively identify ES4 as a chemical tool enabling the study of ARF-GEF-mediated processes, including ARF-GEF-mediated plant development

Itineraries of enzymatically and non-enzymatically catalyzed substitutions at O-glycopyranosidic bonds

Several crystal structures of glycoside hydrolases in complex with a substrate analog, or of inactive mutants complexed with a substrate, reveal non-ground state carbohydrate conformations within subsite -1 of the active site. These "frozen" local minima as preferred by the enzyme represent pre-transition-state situations along the reaction itinerary. Substantiated by theoretical considerations, this leads to the proposal that substitutions on beta-equatorial as well as alpha-axial D-O-glycopyranosidic bonds may always follow an itinerary that is predetermined by the original configuration at the anomeric center, where the formation of a transition state that is similar to a half-chair is always preceded by a conformational change away from the ground state