Sikorra Stefan, Litschko Christa, Müller Carina, Thiel Nadine, Galli Thierry, Eichner Timo, Binz Thomas
Institut für Physiologische Chemie, OE 4310, Medizinische Hochschule Hannover, D-30623 Hannover, Germany.
Sorbonne Paris Cité, Université Paris Diderot, Institut Jacques Monod, CNRS UMR 7592, INSERM ERL U950, F-75013 Paris, France.
J Mol Biol. 2016 Jan 29;428(2 Pt A):372-384. doi: 10.1016/j.jmb.2015.10.024. Epub 2015 Oct 30.
Botulinum neurotoxins (BoNTs) are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs (soluble N-ethyl maleimide sensitive factor attachment protein receptors). However, their serotype A (BoNT/A) that cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved. Therefore, we examined by mutational analyses the reason for the resistance of human SNAP-23, an isoform of SNAP-25. We show that replacement of 10 SNAP-23 residues with their SNAP-25 counterparts effects SNAP-25-like cleavability. Conversely, transfer of each of the replaced SNAP-23 residues to SNAP-25 drastically decreased the cleavability of SNAP-25. By means of the existing SNAP-25-toxin co-crystal structure, molecular dynamics simulations, and corroborative mutagenesis studies, the appropriate binding pockets for these residues in BoNT/A were characterized. Systematic mutagenesis of two major BoNT/A binding pockets was conducted in order to adapt these pockets to corresponding amino acids of human SNAP-23. Human SNAP-23 cleaving mutants were isolated using a newly established yeast-based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on SNAP-23-mediated hypersecretion.
肉毒杆菌神经毒素（BoNTs）是一种高效的细菌蛋白，它通过切割SNAREs（可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体）来阻断神经肌肉接头处的神经递质释放。然而，其切割SNAP-25（25 kDa的突触体相关蛋白）的A型（BoNT/A）在25年来一直是一种成熟的药物，用于治疗依赖胆碱能神经末梢过度活跃的病症。其应用扩展到与分泌过多成分相关的各种其他病症受到了一定阻碍，部分原因是相关的SNARE亚型不会被切割。因此，我们通过突变分析研究了SNAP-25的一种亚型——人类SNAP-23具有抗性的原因。我们发现，将10个SNAP-23残基替换为其对应的SNAP-25残基会影响其类似SNAP-25的可切割性。相反，将每个被替换的SNAP-23残基转移到SNAP-25上会大幅降低SNAP-25的可切割性。借助现有的SNAP-25-毒素共晶体结构、分子动力学模拟以及确证性诱变研究，对BoNT/A中这些残基的合适结合口袋进行了表征。对两个主要的BoNT/A结合口袋进行了系统性诱变，以使这些口袋适应人类SNAP-23的相应氨基酸。使用新建立的基于酵母的筛选系统分离出了能够切割人类SNAP-23的突变体。该方法可能有助于设计新型BoNT/A药物来治疗依赖SNAP-23介导的分泌过多的疾病。
