Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies

Abstract

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.

Yeast Display Enables Identification of Covalent Single Domain Antibodies Against Botulinum Neurotoxin Light Chain A

“…To maximize the probability of identifying ncAA-substituted sdAbs that facilitate irreversible binding, we utilized a set of well-characterized, alpaca-derived sdAbs that target different epitopes of the light chain (LC) of botulinum neurotoxin serotype A1 (BoNT/A), and for which detailed structural binding data was available. 51,52 We reasoned that targeting different regions of LC/A with ncAA-substituted sdAbs would increase the likelihood of identifying sites conducive to covalent bond formation (Figure 1). Based on the available crystallographic data of sdAb-LC/A complexes, 51,52 we identified ncAA substitution sites in four sdAbs at positions likely to result in covalent interaction.…”

“…51,52 We reasoned that targeting different regions of LC/A with ncAA-substituted sdAbs would increase the likelihood of identifying sites conducive to covalent bond formation (Figure 1). Based on the available crystallographic data of sdAb-LC/A complexes, 51,52 we identified ncAA substitution sites in four sdAbs at positions likely to result in covalent interaction. Three of the chosen sdAbs, JPU-A5, JPU-C1, and JPU-C10, are potent inhibitors of LC/A protease activity (Figure 1b, c) 51 and one LC/A-binding sdAb, ciA-H7, has no protease inhibition activity but prevents BoNT/A intoxication of neuronal cells (Figure 1b, c).…”

“…Based on the available crystallographic data of sdAb-LC/A complexes, 51,52 we identified ncAA substitution sites in four sdAbs at positions likely to result in covalent interaction. Three of the chosen sdAbs, JPU-A5, JPU-C1, and JPU-C10, are potent inhibitors of LC/A protease activity (Figure 1b, c) 51 and one LC/A-binding sdAb, ciA-H7, has no protease inhibition activity but prevents BoNT/A intoxication of neuronal cells (Figure 1b, c). 52 Guided by these sdAb-LC/A complex structures, we identified substitution positions in sdAbs that we deemed likely to result in photocrosslinking to LC/A based on 1) our recent developments in studying AzF-mediated photocrosslinking events on the yeast surface; 33 and 2) access to the AcFRS orthogonal translation system (OTS) to introduce this ncAA in response to the TAG codon in S. cerevisiae (Figure 1b, c).…”

“…This in line with the crystallographic data, which shows that interactions between WT JPU C1 and LC/A occur within this domain. 51 With convincing evidence for crosslinking interactions in solution, we next sought to evaluate covalent adduct formation as a function of time. SDS-PAGE analysis facilitated characterization of the formation of the photocrosslinked LC/A-sdAb adduct for varying irradiation times.…”

“…This strongly suggests that both WT and OBeY-containing sdAbs are capable of inhibiting the autoproteolytic activity of LC/A, implying that OBeY substitution does not overtly disrupt the demonstrated inhibitory properties of WT JPU-C1. 51 To quantitatively characterize the inhibitory properties of all three ncAA-sdAb variants considered here, we used a commercial FRET-based LC/A substrate to monitor enzymatic activity over time. By monitoring changes in reporter fluorescence over time in mixtures of a fixed LC/A concentration and varying sdAb amounts, we were able to determine the initial rates of reaction.…”