A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency

Abstract

The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin’s cell binding domain HC, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity.

Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins

“…Functionally, H C determines neuronal specificity by recognizing a polysialoganglioside (e.g., GT1b) and a protein receptor, synaptotagmin (Syt) I/II (for BoNT/B, /G, and /DC) or glycosylated synaptic vesicle protein 2 (SV2) (for BoNT/A, /D, /E, and /F), located on the presynaptic membrane (Chai et al, 2006;Jin et al, 2006;Montecucco, 1986;Stenmark et al, 2008;Yao et al, 2016). H C of BoNT/B, /G, and /DC additionally carries a hydrophobic loop, termed the H Cloop, which interacts with host membrane lipids (Stern et al, 2018;Zhang et al, 2017;Figure 1A). Under acidic conditions, the H N undergoes a pH-induced structural rearrangement and forms a protein channel that delivers the unfolded LC to the cytosol (Fischer et al, 2012;Koriazova and Montal, 2003;Lam et al, 2018;Montal, 2009).…”

Characterization of a membrane binding loop leads to engineering botulinum neurotoxin B with improved therapeutic efficacy

“…The role of LBLs in BoNT/B, C, D, G, and DC for binding to lipid membranes has been hypothesized previously. Mutagenesis studies have demonstrated the importance of LBL for forming stable binding to membrane-embedded receptors and for the potency of these toxins [64][65][66][67][68]71]. However, it has been challenging to detect direct binding of these toxins to lipid membranes because protein−lipid interactions are usually transient and of rather low binding affinity.…”

“…To examine the effect of lipid binding in the presence of Syt II, we compared binding of H C /B versus H C /B WW for ND-Syt. As binding of H C /B to Syt II has a relatively high affinity, with K D approximately 10 −7 M [46,47,71], we resorted to the more-sensitive Octet RED 384 system for BLI assays and titrated the H C concentrations from 2 μM down to 5 nM (Fig 4D-4F). Binding responses, total binding levels, and dissociation rates were all similar between WT H C /B and H C /B WW .…”

“…Indeed, utilizing liposome flotation assays, we recently showed that H C /DC binds directly to liposomes containing only phosphatidylcholine (PC), and mutations at the tip of this extended loop abolish lipid-binding capability, demonstrating that this extended loop, designated "lipid-binding loop" (LBL), mediates membrane binding independent of gangliosides in BoNT/DC [29]. A recent study further examined the role of LBLs in BoNT/B, G, and DC, showing that deleting 3-5 residues in LBL drastically reduced the potency of BoNT/B, G, and DC [71]. Furthermore, H C /B, H C /G, and H C /DC with deletion mutations in their LBLs lost the ability to form stable binding to CGs or Syt II embedded in Triton X-100 micelles or in lipid environment in nanodiscs [71].…”

“…A recent study further examined the role of LBLs in BoNT/B, G, and DC, showing that deleting 3-5 residues in LBL drastically reduced the potency of BoNT/B, G, and DC [71]. Furthermore, H C /B, H C /G, and H C /DC with deletion mutations in their LBLs lost the ability to form stable binding to CGs or Syt II embedded in Triton X-100 micelles or in lipid environment in nanodiscs [71].…”