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

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.肉毒神经毒素（Botulinum neurotoxins, BoNTs）是一类拥有七种主要血清型（BoNT/A至G）的细菌毒素。这类毒素靶向并结合运动神经末梢的能力，是决定其效力与效能的核心因素。在上述毒素中，BoNT/B是获批用于医疗与美容用途的两种血清型之一。除结合已被广泛确认的受体外，有研究提出，BoNT/B的羧基端受体结合结构域（HC，即HC/B）中的延伸环，可通过与脂质膜相互作用辅助毒素结合神经元，该环被称为脂质结合环（LBL）。BoNT/C、D、G以及嵌合毒素DC的HC区中均存在类似的环结构。然而，此前检测并表征LBL与脂质膜的结合特性一直颇具挑战。本研究借助纳米盘系统（nanodisc system）与生物层干涉术（biolayer interferometry）开展检测，结果发现HC/DC、HC/C与HC/G可直接结合无受体脂质，而HC/B与HC/D则无此能力，其中HC/DC的结合水平最高。诱变实验证实，LBL顶端的连续芳香族残基在HC/DC结合脂质膜的过程中发挥关键作用。基于这一机制发现，我们将HC/B的LBL顶端的两个非芳香族残基替换为色氨酸（tryptophan），成功构建了功能获得性（gain-of-function）突变体HC/B。共结晶（cocrystallization）研究证实，这两个色氨酸残基并未改变HC/B的结构，也未影响其与受体的结合相互作用。该突变体HC/B不仅获得了结合无受体脂质膜的能力，对培养神经元的结合能力也得到了增强。最后，我们制备了在LBL中携带两个色氨酸突变、同时携带有两个可增强其与人受体结合能力的额外突变（E1191M/S1199Y）的全长BoNT/B，并通过趾外展评分（Digit Abduction Score, DAS）实验在小鼠体内开展评价。与野生型BoNT/B对照相比，该突变毒素在注射部位局部肌肉麻痹方面的效能有所提升，且全身扩散程度更低，因此既扩大了安全范围，又延长了麻痹持续时间。本研究的发现阐明了LBL与脂质相互作用的机制，并构建了治疗效能得到优化的改造型BoNT/B。