Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX-猫眼课题宝

DOI：

10.1101/2022.01.14.476343

发表时间：

2022-01

期刊：

Proceedings of the National Academy of Sciences of the United States of America

影响因子：

11.1

通讯作者：

中科院分区：

综合性期刊1区

文献类型：

作者：

Lainey J Williamson;M. Galchenkova;Hannah L Best;Richard Bean;A. Munke;S. Awel;Gisel Pena;J. Knoška;R. Schubert;Katerina Dörner;Hyun-Woo Park;D. Bideshi;A. Henkel;V. Kremling;Bjarne Klopprogge;Emyr Lloyd-Evans;Mark T Young;J. Valerio;M. Kloos;M. Sikorski;G. Mills;J. Bielecki;H. Kirkwood;Chan Kim;R. de Wijn;K. Lorenzen;P. L. Xavier;Aida Rahmani Mashhour;L. Gelisio;O. Yefanov;A. Mancuso;B. Federici;Henry N. Chapman;N. Crickmore;P. Rizkallah;Colin Berry;D. Oberthür

来自球形Lysinibacillus sphaericus的Tpp49Aa1是一个Toxin_10家族蛋白，与Cry48Aa1 （3-domain crystal protein）结合后，对致倦库蚊具有较强的杀蚊活性。利用纳米聚焦x射线自由电子激光器的MHz序列飞秒晶体学，以1.62 Å分辨率从天然纳米晶体中快速、高质量地收集数据，确定了Tpp49Aa1的结构。这揭示了Tpp49Aa1在这些从孢子菌中分离出来的纳米晶体中作为具有大分子间界面的同型二聚体的包装，为自然结晶提供了线索。在不同pH值下进行的补充实验也可以研究导致天然Tpp49Aa1晶体溶解的早期结构事件。通过建模，我们提出了Tpp49Aa1和Cry48Aa1之间的潜在相互作用，可能在它们的相互依赖中发挥作用，并拓宽了我们对这个双组分系统的理解。我们扩大了已知的目标范围，证明了斯氏按蚊、白纹伊蚊和tarsalis库蚊的幼虫对Tpp49Aa1/Cry48Aa1的敏感性，从而大大增加了该毒素对在蚊虫控制中的潜在应用。研究人员利用库蚊细胞系进一步了解了Cry48Aa1/Tpp49Aa1细胞模型的功能，为未来研究Cry48Aa1/Tpp49Aa1的作用机制提供了条件，并证明了单个毒素成分的短暂有害影响。Tpp49Aa1/Cry48Aa1蛋白对杀灭蚊子幼虫。创新使用纳米聚焦x射线自由电子激光器，以匹配天然Tpp49Aa1纳米晶体的尺寸和任何XFEL中可用的最高光束强度，用于高通量数据收集，允许结构分辨率达到1.62 Å。Tpp蛋白表现出一系列与不同伙伴的相互作用，从而引发毒性。为了深入了解Tpp49Aa1，对其与Cry48Aa1的相互作用进行了建模。我们还建立了基于细胞的Tpp49Aa1/Cry48Aa1活性测定方法。我们扩大了已知的目标范围，增加了三种蚊子：斯氏按蚊、白纹伊蚊和库蚊。该研究将为未来的Tpp行动模式调查提供基础，并有助于优化针对西尼罗河病毒和疟疾等新发疾病蚊媒的杀虫剂。

Tpp49Aa1 from Lysinibacillus sphaericus is a Toxin_10 family protein that – in combination with Cry48Aa1, a 3-domain crystal protein - has potent mosquitocidal activity, specifically against Culex quinquefasciatus mosquitoes. MHz serial femtosecond crystallography at a nano-focused X-ray free electron laser, allowed rapid and high-quality data collection to determine the Tpp49Aa1 structure at 1.62 Å resolution from native nanocrystals. This revealed the packing of Tpp49Aa1 within these nanocrystals, isolated from sporulated bacteria, as a homodimer with a large intermolecular interface, shedding light on natural crystallization. Complementary experiments conducted at varied pH also enabled investigations of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals. Using modelling, we propose a potential interaction between Tpp49Aa1 and Cry48Aa1 that may play a role in their codependency and broaden our understanding of this two-component system. We expand the known target range, demonstrating Tpp49Aa1/Cry48Aa1 susceptibility of larvae from Anopheles stephensi, Aedes albopictus and Culex tarsalis – substantially increasing the potential use of this toxin pair in mosquito control. Further functional insights are gained using Culex cell lines to characterise cellular models for future investigations into Cry48Aa1/Tpp49Aa1 mechanism of action and to demonstrate transient detrimental effects of individual toxin components. Significance Statement The Tpp49Aa1/Cry48Aa1 protein pair kills mosquito larvae. Innovative use of nano-focused X-ray free electron laser to match the size of natural Tpp49Aa1 nanocrystals and the highest beam intensity available in any XFEL for high-throughput data collection, allowed structural resolution to 1.62 Å. Tpp proteins show a range of interactions with different partners to elicit toxicity. To gain insight into Tpp49Aa1, its interaction with Cry48Aa1 was modelled. We also establish cell-based assays of Tpp49Aa1/Cry48Aa1 activity. We expand the known target range to include three more mosquito species: Anopheles stephensi, Aedes albopictus and Culex tarsalis. This study will underpin future Tpp mode of action investigations and aid insecticide optimization against mosquito vectors of emerging diseases such as West Nile Virus and malaria.

Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX

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