Compared to conventional antisera strategies, monoclonal antibodies (mAbs) represent an alternative and safer way to treat botulism, a fatal flaccid paralysis due to botulinum neurotoxins (BoNTs). In addition, mAbs offer the advantage to be produced in a reproducible manner. We previously identified a unique and potent mouse mAb (TA12) targeting BoNT/A1 with high affinity and neutralizing activity. In this study, we characterized the molecular basis of TA12 neutralization by combining Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) with site-directed mutagenesis and functional studies. We found that TA12 recognizes a conformational epitope located at the interface between the H CN and H CC subdomains of the BoNT/A1 receptor-binding domain (H C ). The TA12-binding interface shares common structural features with the ciA-C2 VHH epitope and lies on the face opposite recognized by ciA-C2-and the CR1/CR2-neutralizing mAbs. The single substitution of N1006 was sufficient to affect TA12 binding to H C confirming the position of the epitope. We further uncovered that the TA12 epitope overlaps with the BoNT/ A1-binding site for both the neuronal cell surface receptor synaptic vesicle glycoprotein 2 isoform C (SV2C) and the GT1b ganglioside. Hence, TA12 potently blocks the entry of BoNT/A1 into neurons by interfering simultaneously with the binding of SV2C and to a lower extent GT1b. Our study reveals the unique neutralization mechanism of TA12 and emphasizes on the potential of using single mAbs for the treatment of botulism type A.
The spontaneous opening of large transendothelial cell macroaperture (TEM) tunnels can accompany leukocyte diapedesis and is triggered by bacterial exoenzymes that inhibit RhoA-driven cytoskeleton contractility. Modelling the dynamics of TEM via a theoretical framework used for soft matter physics allowed us to depict the essential driving forces at play on the membrane to enlarge TEMs. In this study, we conducted multidisciplinary experiments to characterize the role respectively played by cavin-1-structured caveolae and non-caveolar caveolin-1 in plasma membrane mechanics and identify their functional effects on TEM size. The results pointed towards a contributing role for non-caveolar caveolin-1 in the membrane bending rigidity, a mechanical parameter we quantified in a model system of tubes pulled from plasma membrane spheres. Depletion of cavin-1-structured caveolae showed no effect on membrane rigidity, whereas caveolae controlled cell height favouring TEM nucleation. Hence, caveolae confer protection against exoenzyme EDIN-B in mice with staphylococcal septicaemia.
The cytotoxic necrotizing factor 1 (CNF1) toxin from uropathogenic Escherichia coli constitutively activates Rho GTPases by catalyzing the deamidation of a critical glutamine residue located in the switch II (SWII). In crystallographic structures of the CNF1 catalytic domain (CNF1CD), surface-exposed P768 and P968 peptidyl-prolyl imide bonds (X-Pro) adopt an unusual cis conformation. Here, we show that mutation of each proline residue into glycine abrogates CNF1CD in vitro deamidase activity, while mutant forms of CNF1 remain functional on RhoA in cells. Using molecular dynamics simulations coupled to protein-peptide docking, we highlight the long-distance impact of peptidyl-prolyl cis-trans isomerization on the network of interactions between the loops bordering the entrance of the catalytic cleft. The energetically favorable isomerization of P768 compared with P968, induces an enlargement of loop L1 that fosters the invasion of CNF1CD catalytic cleft by a peptide encompassing SWII of RhoA. The connection of the P968 cis isomer to the catalytic cysteine C866 via a ladder of stacking interactions is alleviated along the cis-trans isomerization. Finally, the cis-trans conversion of P768 favors a switch of the thiol side chain of C866 from a resting to an active orientation. The long-distance impact of peptidyl-prolyl cis-trans isomerizations is expected to have implications for target modification.
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