Masuyer Geoffrey, Davies Jonathan R, Moore Kevin, Chaddock John A, Ravi Acharya K
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK.
Current address: Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, 10691 Stockholm, Sweden.
Sci Rep. 2015 Sep 1;5:13397. doi: 10.1038/srep13397.
The botulinum neurotoxin type D is one of seven highly potent toxins produced by Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3 Å, and that of SXN101959 at 3.1 Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D. Taken together, these results demonstrate the robustness of the 'LHn fold' across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.
D型肉毒杆菌神经毒素是肉毒杆菌产生的七种高效毒素之一，可抑制胆碱能神经末梢的神经传递。一种源自该毒素的功能性片段LHn，由催化结构域和转位结构域组成，被誉为开发靶向分泌抑制剂的平台。这些分泌抑制剂旨在将毒素重新靶向特定细胞类型以抑制囊泡分泌。在此，我们报告了D型血清型LHn在2.3 Å分辨率下的晶体结构，以及SXN101959在3.1 Å分辨率下的晶体结构。SXN101959是一种将D型血清型LHn与生长激素释放激素片段结合的衍生物，此前已在抑制垂体生长激素细胞释放生长激素方面显示出有前景的结果。这些结构首次揭示了D型血清型中转位结构域与催化结构域的相互作用。此外，还比较了A、B和D型血清型中LHn/D的小角X射线散射的结构信息。综上所述，这些结果证明了“LHn折叠”在不同血清型中的稳定性及其在工程化具有附加功能的其他多肽组分中的应用。我们的研究证明了肉毒杆菌神经毒素，特别是D型血清型，作为工程化新型分泌抑制剂基础的适用性。
