广泛中和艰难梭菌毒素B变体的迷你蛋白结合剂的从头设计-动脉网

De novo design of mini-protein binders broadly neutralizing Clostridioides difficile toxin B variants

Nature 等信源发布 2024-10-02 16:25

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AbstractClostridioides difficile toxin B (TcdB) is the key virulence factor accounting for C. difficile infection-associated symptoms. Effectively neutralizing different TcdB variants with a universal solution poses a significant challenge. Here we present the de novo design and characterization of pan-specific mini-protein binders against major TcdB subtypes.

摘要艰难梭菌毒素B（TcdB）是导致艰难梭菌感染相关症状的关键毒力因子。用通用解决方案有效中和不同的TcdB变体是一项重大挑战。在这里，我们介绍了针对主要TcdB亚型的泛特异性小蛋白结合剂的从头设计和表征。

Our design successfully binds to the first receptor binding interface (RBI-1) of the varied TcdB subtypes, exhibiting affinities ranging from 20 pM to 10 nM. The cryo-electron microscopy (cryo-EM) structures of the mini protein binder in complex with TcdB1 and TcdB4 are consistent with the computational design models.

我们的设计成功地结合了各种TcdB亚型的第一个受体结合界面（RBI-1），表现出从20pM到10nM的亲和力。与TcdB1和TcdB4复合的微型蛋白质粘合剂的低温电子显微镜（cryo-EM）结构与计算设计模型一致。

The engineered and evolved variants of the mini-protein binder and chondroitin sulfate proteoglycan 4 (CSPG4), another natural receptor that binds to the second RBI (RBI-2) of TcdB, better neutralize major TcdB variants both in cells and in vivo, as demonstrated by the colon-loop assay using female mice.

迷你蛋白粘合剂和硫酸软骨素蛋白聚糖4（CSPG4）的工程化和进化变体是另一种与TcdB的第二个RBI（RBI-2）结合的天然受体，可以更好地中和细胞和体内的主要TcdB变体，如使用雌性小鼠的结肠环测定所证明的。

Our findings provide valuable starting points for the development of therapeutics targeting C. difficile infections (CDI)..

我们的研究结果为开发针对艰难梭菌感染（CDI）的治疗药物提供了有价值的起点。。

IntroductionClostridioides difficile (also known as Clostridium difficile) is an opportunistic pathogen that mainly infects human colons, leading to the breakdown of the colonic epithelial barrier1,2. This, in turn, results in diarrhea, colitis, and even gastroenteritis-associated death. CDI is responsible for approximately half a million cases (mostly nosocomial and community-acquired) and 15,000 deaths each year in the United States3,4.

引言艰难梭菌（Clostridioides difficile，也称艰难梭菌）是一种机会性病原体，主要感染人类结肠，导致结肠上皮屏障的破坏1,2。这反过来会导致腹泻，结肠炎，甚至与胃肠炎相关的死亡。CDI每年在美国造成约50万例（主要是医院和社区获得）和15000例死亡3,4。

The pathogenic C. difficile secretes one or more of the three exotoxins, including toxin A (TcdA), toxin B (TcdB), and C. difficile transferase (CDT), to cause intestinal injuries5. Of the three toxins, TcdB is required for the high virulence of pathogenic strains in animal models, and TcdB alone is sufficient to induce a wide range of diseases in humans6,7,8,9.TcdB is composed of multiple domains with a molecular weight of ~270 kDa.

致病性艰难梭菌分泌三种外毒素中的一种或多种，包括毒素A（TcdA），毒素B（TcdB）和艰难梭菌转移酶（CDT），以引起肠道损伤5。在这三种毒素中，TcdB是动物模型中致病菌株高毒力所必需的，单独的TcdB足以在人类中诱发多种疾病6,7,8,9.TcdB由分子量为〜270 kDa的多个结构域组成。

TcdB consists of an N-terminal glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a delivery and receptor-binding domain (DRBD), and a large C-terminal combined repetitive oligopeptides domain (CROPs) (Supplementary Fig. 1). TcdB utilizes the GTD to glucosylate small guanosine triphosphatase (GTPase) proteins, thereby disrupting the cytoskeleton and gradually causing cell death10,11,12.Natural variants of TcdB are commonly seen in the clinics13,14.

TcdB由N端葡萄糖基转移酶结构域（GTD），半胱氨酸蛋白酶结构域（CPD），递送和受体结合结构域（DRBD）和大的C端组合重复寡肽结构域（CROPs）组成（补充图1）。TcdB利用GTD将小的鸟苷三磷酸酶（GTPase）蛋白糖基化，从而破坏细胞骨架并逐渐导致细胞死亡10,11,12。TcdB的自然变异在临床上很常见13,14。

Based on recent studies, TcdB can be divided into at least eight subtypes, while four of them (known as TcdB1–4) are closely associated with human diseases15,16. These TcdB variants recognize varied host receptors through two separate interfaces (Supplementary Fig. 1)17,18. The first receptor-binding interface (RBI-1) is located at the convex edge of the DRBD: TcdB1 and TcdB3 bind Frizzled proteins (FZDs)19,20,21,22, while TcdB2 and TcdB4, which.

根据最近的研究，TcdB可分为至少八种亚型，其中四种（称为TcdB1-4）与人类疾病密切相关15,16。这些TcdB变体通过两个单独的界面识别不同的宿主受体（补充图1）17,18。第一个受体结合界面（RBI-1）位于DRBD的凸边缘：TcdB1和TcdB3结合卷曲蛋白（FZDs）19,20,21,22，而TcdB2和TcdB4则结合。

For the complex structure of TcdB1-minibinder

对于TcdB1微型粘合剂的复杂结构

The initial structure model of the mini-protein binder was imported from the design model. This was combined with the structure of the TcdB1 DRBD domain (residues 1300–1800, PDB code: 6C0B) and the structure of TcdB1 (PDB code: 7ML7), which includes the GTD domain, CPD domain, and parts of the CROPs domain.

从设计模型中导入了微型蛋白质粘合剂的初始结构模型。。

These models were fitted into the cryo-EM map and combined using UCSF Chimera and Coot, followed by iterative rounds of real-space refinement in PHENIX, cartesian and torsion adjustments using RosettaCM, and further manual adjustments in COOT54,55,65..

这些模型被拟合到cryo-EM图中，并使用UCSF Chimera和Coot进行组合，然后使用RosettaCM在PHENIX中进行迭代轮的真实空间优化，笛卡尔和扭转调整，并在COOT54,55,65中进行进一步的手动调整。。

For building the model of TcdB4-minibinder

用于建立TcdB4 minibinder的模型

The initial structure model for TcdB4 was imported from the TcdB4-TFPI complex (PDB code: 7V1N), and the initial minibinder structure model was generated by Rosetta in the design phase. The model was fitted into the map by the dock_in_map process in PHENIX65. Like the TcdB1-minibinder complex, iterative rounds of adjustments were made through real-space refinement, RosettaCM, and manual operations in COOT54,55,65..

TcdB4的初始结构模型是从TcdB4 TFPI复合体（PDB代码：7V1N）导入的，初始minibinder结构模型是由Rosetta在设计阶段生成的。该模型通过PHENIX65中的dock\u in\u map过程拟合到地图中。像TcdB1 minibinder complex一样，通过实空间优化，RosettaCM和COOT54,55,65中的手动操作进行了反复的调整。。

The complex structures of TcdB1-minibinder and TcdB4-minibinder were validated by examining the Molprobity scores and analyzing the statistics of the Ramachandran plots66. Please refer to Supplementary Table 2 for detailed statistics on the 3D reconstructions and model refinement. All structure figures were prepared by using PyMOL (https://www.pymol.org) and UCSF ChimeraX67..

通过检查Molprobity评分和分析Ramachandran图的统计数据，验证了TcdB1 minibinder和TcdB4 minibinder的复杂结构66。有关3D重建和模型改进的详细统计信息，请参阅补充表2。所有结构图均使用PyMOL制备(https://www.pymol.org)和UCSF ChimeraX67。。

Cytopathic cell-rounding assayThe cytopathic effect (cell-rounding) of TcdB was analyzed using the gold-standard cell-rounding assay. The cells were incubated with a mixture of toxin and inhibitor in different proportions in vitro, following the addition of a complete medium. The cells were photographed four to six hours after they were incubated.

细胞病变细胞圆形测定使用金标准细胞圆形测定法分析TcdB的细胞病变效应（细胞圆形）。加入完全培养基后，将细胞与不同比例的毒素和抑制剂混合物在体外孵育。孵育后4至6小时对细胞拍照。

10×–20× objectives on an Olympus IX73 microscope equipment were used to get the phase-contrast pictures. A zone of 300 × 300 μm was selected randomly, which contains 20~100 cells. Round-shaped and normal-shaped cells were counted manually, and the percentage of round-shaped cells was calculated.Mouse cytokine assayMice were injected intraperitoneally with a certain amount of Fc_H3_5N2 or PBS.

使用Olympus IX73显微镜设备上的10×–20×物镜获得相衬图像。随机选择300×300μm的区域，其中包含20〜100个细胞。手动计数圆形和正常形状的细胞，并计算圆形细胞的百分比。小鼠细胞因子测定小鼠腹膜内注射一定量的Fc\u H3\u 5N2或PBS。

Whole blood was collected from the heart 2 hours or 48 hours later. The blood samples were centrifuged at 4 °C for 15 minutes, and the supernatant, which is the serum, was collected. Cytokines in the serum samples were measured using the BD cytometric bead array and Mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences, CA, USA).

2小时或48小时后从心脏收集全血。将血样在4℃下离心15分钟，收集上清液，即血清。使用BD细胞计数珠阵列和小鼠Th1/Th2/Th17细胞因子试剂盒（BD Biosciences，CA，USA）测量血清样品中的细胞因子。

According to the manufacturer’s instructions, we tested the cytokines, including mouse IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF. 50 μL of the mixed captured beads, 50 μL of the serum sample or standard dilutions, and 50 μL of phycoerythrin (PE) detection reagent were added consecutively to each assay tube and incubated for 2 hours at room temperature in the dark.

。将50μL混合捕获的珠子，50μL血清样品或标准稀释液和50μL藻红蛋白（PE）检测试剂连续加入每个测定管中，并在室温下在黑暗中孵育2小时。

The samples were measured on the Beckman Flow Cytometer CytoFLEX LX-5L1 and analyzed using FCAP Array Software (BD Biosciences).Evaluation of the stability of the binderAfter the mice were euthanized, the colon segments were excised and homogenized. The homogenate was then centrifuged, and the supernatant was collected.

在Beckman流式细胞仪CytoFLEX LX-5L1上测量样品，并使用FCAP阵列软件（BD Biosciences）进行分析。粘合剂稳定性的评估小鼠安乐死后，切除结肠段并匀浆。然后离心匀浆，收集上清液。

The supernatant was incubated with the bi.

将上清液与bi一起温育。

Data availability

数据可用性

The cryo-EM maps have been deposited in the Electron Microscopy Data Bank under accession number EMD-39072 (TcdB1 in complex with mini-protein binder), EMD-39073 (TcdB4 in complex with mini-protein binder) with local refinement map provided as additional map, and the associated models have been deposited in the RSCB Protein Data Bank under accession number 8Y9B (TcdB1 in complex with mini-protein binder), 8Y9C (TcdB4 in complex with mini-protein binder).

冷冻电镜图谱已保存在电子显微镜数据库中，登录号为EMD-39072（TcdB1与迷你蛋白粘合剂复合），EMD-39073（TcdB4与迷你蛋白粘合剂复合），局部细化图谱作为附加图谱提供，相关模型已保存在RSCB蛋白质数据库中，登录号为8Y9B（TcdB1与迷你蛋白粘合剂复合），8Y9C（TcdB4与迷你蛋白粘合剂复合）。

The raw data of DNA high-throughput sequencing is submitted to NCBI SRA in the BioProject PRJNA1154555, and the processed data are provided in FigShare [https://doi.org/10.6084/m9.figshare.26562841]. Source data are provided with this paper..

DNA高通量测序的原始数据已提交给生物项目PRJNA1154555中的NCBI SRA，处理后的数据在FigShare中提供[https://doi.org/10.6084/m9.figshare.26562841]。本文提供了源数据。。

Code availability

代码可用性

The data that support this study and Rosetta scripts are available as source data provided in Figshare, under the accession code [https://doi.org/10.6084/m9.figshare.26562841]. Rosetta Modeling Suit 2019.47.61047 (https://www.rosettacommons.org/) is available to academic and non-commercial users for free.

支持这项研究的数据和Rosetta脚本可以作为Figshare提供的源数据，根据加入代码[https://doi.org/10.6084/m9.figshare.26562841]。Rosetta模特套装2019.47.61047(https://www.rosettacommons.org/)免费提供给学术和非商业用户。

The source code for RIF docking is available at https://github.com/rifdock/rifdock. Binding data was collected in Octet RED96 and processed using Octet Analysis software. Flow cytometry data was collected in Beckman Flow Cytometer CytoFLEX LX-5L and BD FACSMelody™ Cell Sorter and processed by Flowjo v9.

RIF对接的源代码可在https://github.com/rifdock/rifdock.在Octet RED96中收集结合数据，并使用Octet分析软件进行处理。。

The figures were generated by Adobe Illustrator. The cryo-EM data are processed by CryoSPARC 4.2.1, Coot-0.9.5 and Phenix-1.19.2..

这些数字是由Adobe Illustrator生成的。cryo-EM数据由CryoSPARC 4.2.1，Coot-0.9.5和Phenix-1.19.2处理。。

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Download referencesAcknowledgementsWe would like to thank the flow cytometry facility, microscopy facility, cryo-EM facility, the Westlake University HPC Center for computation assistance, and the Protein Characterization and Crystallography Facility of Westlake University for help in sample analysis.

下载参考文献致谢我们要感谢流式细胞仪设施，显微镜设施，cryo-EM设施，西湖大学HPC计算辅助中心以及西湖大学蛋白质表征和晶体学设施在样品分析方面的帮助。

This work was funded by the Ministry of Science and Technology of the People’s Republic of China project 2020YFA0909200 (P.L.) and project 2023YFC2308403 (L.T.), “Pioneer” and “Leading Goose” R&D Program of Zhejiang (Grant no. 2024SSYS0036), Zhejiang Provincial Natural Science Foundation of China Grant No.

这项工作由中华人民共和国科学技术部项目2020YFA0909200（P.L.）和项目2023YFC2308403（L.T.）资助，浙江省“先锋”和“领雁”研发计划（批准号2024SSYS0036），浙江省自然科学基金批准号。

LR23C050001, National Natural Science Foundation of China project 22137005 (P.L.), project 32430002 (L.T.), and project 32301219 (X.L.), Westlake University-Muyuan Joint Research Institute program WU2024MY001 (L.T.), Westlake Center for Genome Editing project 21200000A992210 (L.T.), and the Research Center for Industries of the Future (RCIF) at Westlake University.Author informationAuthor notesThese authors contributed equally: Xinchen Lv, Yuanyuan Zhang.Authors and AffiliationsResearch Center for Industries of the Future, Westlake University, Hangzhou, Zhejiang, 310024, ChinaXinchen Lv, Jianhua Luo, Liang Tao & Peilong LuWestlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, 310024, ChinaXinchen Lv, Jianhua Luo, Liang Tao & Peilong LuKey Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, 310024, ChinaXinchen Lv, Yuanyuan Zhang, Ke Sun, Qi Yang, Jianhua Luo, Liang Tao & Peilong LuInstitute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, 310024, ChinaXinchen Lv, Ke Sun & Peilong LuCenter for Infectious Disease Research, School of Medicine, Westla.

LR23C050001、国家自然科学基金项目22137005（P.L.）、项目32430002（L.T.）和项目32301219（X.L.）、西湖大学牧源联合研究所项目WU2024MY001（L.T.）、西湖基因组编辑中心项目21200000A992210（L.T.）和西湖大学未来产业研究中心（RCIF）。作者信息作者注意到这些作者做出了同样的贡献：吕新晨，张元元。作者和所属单位西湖大学未来产业研究中心，浙江杭州，310024，中国Lv，罗建华，梁涛和裴隆路西湖生命科学与生物医学实验室，浙江杭州，310024，中国Lv，罗建华，梁涛和裴隆路基浙江省结构生物学实验室，西湖大学生命科学学院，浙江杭州，310024，中国Lv，张元元，孙科，祁阳，罗建华，梁涛和裴隆路基生物研究所，西湖高等研究所，浙江杭州，310024 Lv，Ke Sun和Peilong LuCenter，Westla医学院传染病研究中心。

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PubMed Google ScholarLiang TaoView author publicationsYou can also search for this author in

PubMed Google ScholarLiang TaoView作者出版物您也可以在

PubMed Google ScholarPeilong LuView author publicationsYou can also search for this author in

PubMed Google ScholarPeilong LuView作者出版物您也可以在

PubMed Google ScholarContributionsP.L. and L.T. conceived and supervised the project; X.L. and Y.Z. contributed equally to this work; X.L. designed the minibinder, performed directed evolution, solved the cryo-EM structure with the assistance of K.S.. Y.Z. performed the cell-rounding assay and the colon-loop assay with the help of Q.Y.

PubMed谷歌学术贡献SP。五十、 L.T.构思并监督了该项目；十、 L.和Y.Z.对这项工作做出了同样的贡献；十、 L.设计了微型粘合剂，进行了定向进化，在K.S.的帮助下解决了低温电磁结构。Y.Z.在Q.Y的帮助下进行了细胞圆形测定和结肠环测定。

and J.L.. P.L. and X.L. wrote the original draft and all authors participated in manuscript revision.Corresponding authorsCorrespondence to.

J.L.P.L.和X.L.撰写了原始草案，所有作者都参与了稿件修订。通讯作者通讯。

Liang Tao or Peilong Lu.Ethics declarations

梁涛或陆培龙。道德宣言

Competing interests

相互竞争的利益

X.L., Y.Z., L.T., and P.L. are listed as inventors on a patent application submitted by Westlake University to protect the intellectual property rights of the TcdB toxin binders developed in this study. The remaining authors declare no competing interests.

十、 L.，Y.Z.，L.T。和P.L.被列为西湖大学提交的专利申请的发明人，以保护本研究中开发的TcdB毒素粘合剂的知识产权。其余作者声明没有利益冲突。

Peer review

同行评审

Peer review information

同行评审信息

Nature Communications thanks Amedee des Georges, Timothy Jenkins, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Nature Communications感谢Amedee des Georges，Timothy Jenkins和另一位匿名审稿人对这项工作的同行评审做出的贡献。同行评审文件可用。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary informationSupplementary InformationPeer Review FileReporting SummarySource dataSource DataRights and permissions

。补充信息补充信息同行评审文件报告摘要源数据源数据权限

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material.

开放获取本文是根据知识共享署名非商业性NoDerivatives 4.0国际许可证授权的，该许可证允许以任何媒介或格式进行任何非商业性使用，共享，分发和复制，只要您对原始作者和来源给予适当的信任，提供知识共享许可证的链接，并指出您是否修改了许可材料。

You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/..

要查看此许可证的副本，请访问http://creativecommons.org/licenses/by-nc-nd/4.0/..

Reprints and permissionsAbout this articleCite this articleLv, X., Zhang, Y., Sun, K. et al. De novo design of mini-protein binders broadly neutralizing Clostridioides difficile toxin B variants.

转载和许可本文引用本文Lv，X.，Zhang，Y.，Sun，K。等人。从头设计广泛中和艰难梭菌毒素B变体的微型蛋白质结合剂。

Nat Commun 15, 8521 (2024). https://doi.org/10.1038/s41467-024-52582-1Download citationReceived: 27 February 2024Accepted: 12 September 2024Published: 02 October 2024DOI: https://doi.org/10.1038/s41467-024-52582-1Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

《国家公社》158521（2024）。https://doi.org/10.1038/s41467-024-52582-1Download引文接收日期：2024年2月27日接受日期：2024年9月12日发布日期：2024年10月2日OI：https://doi.org/10.1038/s41467-024-52582-1Share本文与您共享以下链接的任何人都可以阅读此内容：获取可共享链接对不起，本文目前没有可共享的链接。复制到剪贴板。

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