Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493, Japan.
Biochem Biophys Res Commun. 2012 Aug 24;425(2):256-60. doi: 10.1016/j.bbrc.2012.07.077. Epub 2012 Jul 22.
In cell culture supernatants, the botulinum neurotoxin (BoNT) exists as part of a toxin complex (TC) in which nontoxic nonhemagglutinin (NTNHA) and/or hemagglutinins (HAs) are assembled onto the BoNT. A series of investigations indicated that formation of the TC is vital for delivery of the toxin to nerve cells through the digestive tract. In the assembly process, BoNT binds to NTNHA yielding M-TC, and it then matures into L-TC by further association with the HAs via NTNHA in the M-TC. Here, we report a crystal structure of the NTNHA from Clostridium botulinum serotype D strain 4947. Additionally, we performed small-angle X-ray scattering (SAXS) analysis of the NTNHA and the M-TC to elucidate the solution structure. The crystal structure of D-4947 NTNHA revealed that BoNT and NTNHA share a closely related structure consisting of three domains. The SAXS image indicated that, even though the N-terminal two-thirds of the NTNHA molecule had an apparently similar conformation in both the crystal and solution structures, the C-terminal third of the molecule showed a more extended structure in the SAXS image than that seen in the crystallographic image. The discrepancy between the crystal and solution structures implies a high flexibility of the C-terminal third domain of NTNHA, which is involved in binding to BoNT. Structural dynamics of the NTNHA molecule revealed by SAXS may explain its binding to BoNT to form the BoNT/NTNHA complex.
在细胞培养上清液中，肉毒神经毒素（BoNT）作为毒素复合物（TC）的一部分存在，其中非毒性非血凝素（NTNHA）和/或血凝素（HAs）组装到 BoNT 上。一系列研究表明，TC 的形成对于通过消化道将毒素递送到神经细胞至关重要。在组装过程中，BoNT 与 NTNHA 结合生成 M-TC，然后通过与 M-TC 中的 NTNHA 进一步结合，进一步成熟为 L-TC。在这里，我们报告了来自 C. botulinum 血清型 D 菌株 4947 的 NTNHA 的晶体结构。此外，我们还对 NTNHA 和 M-TC 进行了小角度 X 射线散射（SAXS）分析，以阐明其溶液结构。D-4947 NTNHA 的晶体结构表明，BoNT 和 NTNHA 具有密切相关的结构，由三个结构域组成。SAXS 图像表明，尽管 NTNHA 分子的 N 端三分之二在晶体和溶液结构中具有明显相似的构象，但分子的 C 端三分之一在 SAXS 图像中显示出比晶体图像中更伸展的结构。晶体和溶液结构之间的差异表明，NTNHA 分子的 C 端三分之二结构域具有高度的灵活性，这与与 BoNT 的结合有关。SAXS 揭示的 NTNHA 分子的结构动力学可能解释了其与 BoNT 结合形成 BoNT/NTNHA 复合物的原因。
