6QNS

Crystal structure of the binding domain of Botulinum Neurotoxin type B mutant I1248W/V1249W in complex with human synaptotagmin 1 and GD1a receptors

6QNS の概要

• エントリーDOI: 10.2210/pdb6qns/pdb
• 分子名称: Botulinum neurotoxin type B, Synaptotagmin-1, N-acetyl-alpha-neuraminic acid-(2-3)-beta-D-galactopyranose-(1-3)-2-acetamido-2-deoxy-beta-D-galactopyranose-(1-4)-[N-acetyl-alpha-neuraminic acid-(2-3)]beta-D-galactopyranose-(1-4)-beta-D-glucopyranose, ... (4 entities in total)
• 機能のキーワード: botulinum toxin, toxin
• 由来する生物種: Clostridium botulinum; 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 56948.79

構造登録者

Masuyer, G.,Yin, L.,Zhang, S.,Miyashita, S.I.,Dong, M.,Stenmark, P. (登録日: 2019-02-12, 公開日: 2020-02-26, 最終更新日: 2024-01-24)

主引用文献

Yin, L.,Masuyer, G.,Zhang, S.,Zhang, J.,Miyashita, S.I.,Burgin, D.,Lovelock, L.,Coker, S.F.,Fu, T.M.,Stenmark, P.,Dong, M.
Characterization of a membrane binding loop leads to engineering botulinum neurotoxin B with improved therapeutic efficacy.
Plos Biol., 18:e3000618-e3000618, 2020

PubMed Abstract: Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A-G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a "gain-of-function" mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL-lipid interactions and create a modified BoNT/B with improved therapeutic efficacy.

PubMed: 32182233
DOI: 10.1371/journal.pbio.3000618

実験手法

X-RAY DIFFRACTION (2.4 Å)