Nat. Commun：BAM-SurA复合物介导的外膜蛋白组装-动脉网

Nat. Commun：Outer membrane protein assembly mediated by BAM-SurA complexes

Nature 等信源发布 2024-09-01 03:28

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AbstractThe outer membrane is a formidable barrier that protects Gram-negative bacteria against environmental threats. Its integrity requires the correct folding and insertion of outer membrane proteins (OMPs) by the membrane-embedded β-barrel assembly machinery (BAM). Unfolded OMPs are delivered to BAM by the periplasmic chaperone SurA, but how SurA and BAM work together to ensure successful OMP delivery and folding remains unclear.

摘要外膜是保护革兰氏阴性菌免受环境威胁的强大屏障。其完整性需要通过膜嵌入的β-桶组装机器（BAM）正确折叠和插入外膜蛋白（OMP）。未折叠的OMP通过周质伴侣SurA传递给BAM，但SurA和BAM如何协同工作以确保OMP的成功传递和折叠仍不清楚。

Here, guided by AlphaFold2 models, we use disulphide bond engineering in an attempt to trap SurA in the act of OMP delivery to BAM, and solve cryoEM structures of a series of complexes. The results suggest that SurA binds BAM at its soluble POTRA-1 domain, which may trigger conformational changes in both BAM and SurA that enable transfer of the unfolded OMP to the BAM lateral gate for insertion into the outer membrane.

在这里，在AlphaFold2模型的指导下，我们使用二硫键工程试图在OMP传递到BAM的过程中捕获SurA，并解决一系列复合物的cryoEM结构。结果表明，SurA在其可溶性POTRA-1结构域结合BAM，这可能触发BAM和SurA的构象变化，从而使未折叠的OMP转移到BAM侧门以插入外膜。

Mutations that disrupt the interaction between BAM and SurA result in outer membrane assembly defects, supporting the key role of SurA in outer membrane biogenesis..

破坏BAM和SurA之间相互作用的突变会导致外膜组装缺陷，从而支持SurA在外膜生物发生中的关键作用。。

IntroductionGram-negative (diderm) bacteria have a complex outer membrane (OM) that is essential for cell integrity, virulence and pathogenesis1. The OM is densely packed with integral outer membrane proteins (OMPs) that share a β-barrel fold2. These OMPs are synthesised in the cytoplasm, and secreted into the periplasm via Sec-mediated translocation.

引言革兰氏阴性（diderm）细菌具有复杂的外膜（OM），对细胞完整性，毒力和致病性至关重要1。OM中密集堆积着完整的外膜蛋白（OMP），它们共享一个β桶折叠2。这些OMP在细胞质中合成，并通过Sec介导的易位分泌到周质中。

Within the periplasm, they are bound by chaperones, especially Skp and SurA3, before being delivered to the β-barrel assembly machinery (BAM). BAM is an OM-localized, multi-protein complex which folds and inserts OMPs into the OM. The importance of this pathway is underlined by the widespread conservation of the BAM complex in all bacteria that contain a canonical OM, and conservation of both BAM and SurA in proteobacteria4,5.

在周质内，它们被伴侣结合，特别是Skp和SurA3，然后被传递到β-桶组装机器（BAM）。BAM是一种OM定位的多蛋白复合物，可将OMP折叠并插入OM中。BAM复合物在所有含有规范OM的细菌中的广泛保守性以及BAM和SurA在proteobacteria4,5中的保守性强调了这一途径的重要性。

The major component of the BAM complex, BamA, is itself an OMP that is essential in E. coli. BamA is composed of a 16-stranded β-barrel, which, in E. coli, is preceded N-terminally by five polypeptide transport associated (POTRA) domains that extend into the periplasm (Supplementary Fig 1a). BAM also contains four accessory lipoproteins, BamB-E, of which only BamD is essential in E.

BAM复合物的主要成分BamA本身是大肠杆菌中必不可少的OMP。BamA由一个16链的β桶组成，在大肠杆菌中，它在N端之前有五个延伸到周质的多肽转运相关（POTRA）结构域（补充图1a）。BAM还含有四种辅助脂蛋白BamB-E，其中只有BamD在E中是必需的。

coli, and deletions of the other lipoproteins result in growth defects of varying severity6,7. The deletion of SurA also causes a variety of growth defects in an array of pathogenic and lab strains of E. coli in both aerobic and anaerobic conditions8. While structural studies have revealed how BamA inserts OMPs into the OM, the details of how chaperones deliver unfolded OMPs to BAM to begin insertion/assembly remain unknown.

大肠杆菌，其他脂蛋白的缺失导致不同程度的生长缺陷6,7。在需氧和厌氧条件下，SurA的缺失还导致一系列致病性和实验室大肠杆菌菌株的各种生长缺陷8。虽然结构研究揭示了BamA如何将OMP插入OM，但伴侣如何将未折叠的OMP传递给BAM以开始插入/组装的细节仍然未知。

This is especially intriguing given the lack of ATP in the periplasm, which means that these activities cannot be coordinated via ATP binding/hydrolysis, as is commonly used to control and coordinate .

鉴于周质中缺乏ATP，这尤其有趣，这意味着这些活性不能通过ATP结合/水解来协调，这通常用于控制和协调。

Data availability

数据可用性

CryoEM reconstructions and corresponding coordinates have been deposited in the Electron Microscopy Data Bank (EMBD) and the Protein Data Bank (PDB), respectively: Wait Complex Extended SurA (https://www.ebi.ac.uk/emdb/EMD-18035/, https://www.wwpdb.org/pdb?id=pdb_00008pz2), Wait Complex Compact SurA (https://www.ebi.ac.uk/emdb/EMD-18034/, https://www.wwpdb.org/pdb?id=pdb_00008pz1), Wait Complex with Darobactin Extended SurA (https://www.ebi.ac.uk/emdb/EMD-18046/, https://www.wwpdb.org/pdb?id=pdb_00008pzv), Wait Complex with Darobactin Compact SurA (https://www.ebi.ac.uk/emdb/EMD-18045/, https://www.wwpdb.org/pdb?id=pdb_00008pzu), Arrival Complex (https://www.ebi.ac.uk/emdb/EMD-18564/, https://www.wwpdb.org/pdb?id=pdb_00008qpw), Handover Complex (https://www.ebi.ac.uk/emdb/EMD-18563/, https://www.wwpdb.org/pdb?id=pdb_00008qpv), Release Complex Extended SurA (https://www.ebi.ac.uk/emdb/EMD-18543/, https://www.wwpdb.org/pdb?id=pdb_00008qp5), Release Complex Compact SurA (https://www.ebi.ac.uk/emdb/EMD-18053/, https://www.wwpdb.org/pdb?id=pdb_00008q0g) and Release Complex No SurA (https://www.ebi.ac.uk/emdb/EMD-18562/, https://www.wwpdb.org/pdb?id=pdb_00008qpu).

CryoEM重建和相应的坐标已分别保存在电子显微镜数据库（EMBD）和蛋白质数据库（PDB）中：Wait Complex Extended SurA(https://www.ebi.ac.uk/emdb/EMD-18035/，https://www.wwpdb.org/pdb?id=pdb_00008pz2)，Wait Complex Compact苏拉(https://www.ebi.ac.uk/emdb/EMD-18034/，https://www.wwpdb.org/pdb?id=pdb_00008pz1)，Wait复合物与Darobactin扩展的SurA(https://www.ebi.ac.uk/emdb/EMD-18046/，https://www.wwpdb.org/pdb?id=pdb_00008pzv)，Wait复合物与Darobactin Compact SurA(https://www.ebi.ac.uk/emdb/EMD-18045/，https://www.wwpdb.org/pdb?id=pdb_00008pzu)(https://www.ebi.ac.uk/emdb/EMD-18564/，https://www.wwpdb.org/pdb?id=pdb_00008qpw)，移交综合体(https://www.ebi.ac.uk/emdb/EMD-18563/，https://www.wwpdb.org/pdb?id=pdb_00008qpv)，释放复杂的扩展SurA(https://www.ebi.ac.uk/emdb/EMD-18543/，https://www.wwpdb.org/pdb?id=pdb_00008qp5)，发布Complex Compact SurA(https://www.ebi.ac.uk/emdb/EMD-18053/，https://www.wwpdb.org/pdb?id=pdb_00008q0g)并释放复合物No SurA(https://www.ebi.ac.uk/emdb/EMD-18562/，https://www.wwpdb.org/pdb?id=pdb_00008qpu)。

The raw cryoEM datasets used in this study have been deposited to the Electron Microscopy Public Image Archive (EMPIAR): POTRA-1 Cross-link (https://www.ebi.ac.uk/empiar/EMPIAR-12197/), POTRA-1 Cross-link with darobactin (https://www.ebi.ac.uk/empiar/EMPIAR-11933/), POTRA1 Cross-link OmpX hybrid (https://www.ebi.ac.uk/empiar/EMPIAR-11939/), β1 Cross-link OmpX Hybrid (https://www.ebi.ac.uk/empiar/EMPIAR-11940/) and β1 Cross-link EspP Hybrid (https://www.ebi.ac.uk/empiar/EMPIAR-11941/).

Details of all deposition codes are also available in Supplementary Table 2. The raw proteomics data have been deposited to the Prote.

补充表2中还提供了所有沉积代码的详细信息。原始蛋白质组学数据已保存到Prote。

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Download referencesAcknowledgementsWe thank Bob Schiffrin, David Brockwell, and all members of the Radford and Ranson labs for helpful discussions, and Nasir Khan for excellent technical support. CryoEM data were collected at the Astbury Biostructure Laboratory, funded by the University of Leeds and Wellcome (108466/Z/15/Z; 221524/Z/20/Z) and we thank facility staff for their technical input.

下载参考文献致谢我们感谢Bob Schiffrin，David Brockwell以及Radford和Ranson实验室的所有成员进行了有益的讨论，并感谢Nasir Khan提供了出色的技术支持。CryoEM数据是在利兹大学和惠康大学资助的Astbury生物结构实验室收集的（108466/Z/15/Z；221524/Z/20/Z），我们感谢设施工作人员的技术投入。

The Mass Spectrometry Facility instrumentation used in this work was funded by Wellcome (223810/Z/21/Z). K.L.F. and J.E.H. acknowledge funding from the MRC (MR/P018491/1). K.L.F. is funded by the BBSRC (BB/X015653/1) and JAC by BBSRC (BB/T008059/1). SER holds a Royal Society Professorial Fellowship (RSRP/R1/211057).

这项工作中使用的质谱仪器由Wellcome（223810/Z/21/Z）资助。K、 L.F.和J.E.H.感谢MRC的资助（MR/P018491/1）。K、 L.F.由BBSRC（BB/X015653/1）资助，JAC由BBSRC（BB/T008059/1）资助。SER拥有皇家学会教授奖学金（RSRP/R1/211057）。

A.N.C. acknowledges the support of a Sir Henry Dale Fellowship jointly funded by Wellcome and the Royal Society (220628/Z/20/Z) and a University Academic Fellowship from the University of Leeds. T.F.S. and N.B. acknowledge funding from the German Federal Ministry of Education and Research (BMBF, via grant GBi2S and German Centre for Infection Research (DZIF) 09.918).

A、 N.C.感谢惠康（Wellcome）和皇家学会（Royal Society）共同资助的亨利·戴尔爵士（Sir Henry Dale）奖学金（220628/Z/20/Z）和利兹大学（University of Leeds）的大学学术奖学金的支持。T、 F.S.和N.B.感谢德国联邦教育和研究部（BMBF）通过拨款GBi2S和德国感染研究中心（DZIF）09.918提供的资金。

The anti-BamA polyclonal antibody used for western blotting and pTrc99a-BamABCDE-CT8His plasmid was a kind gift from Harris Bernstein (NIH, USA). The anti-SurA polyclonal used for western blotting was a generous gift from Jean-François Collet (UCLouvain, Belgium). The BamA depletion strain (JCM166), SurA deletion strain (AR208), and BamA complementation vector (pZS21-BamA-NT6His) were generously provided by Tom Silhavy (Princeton, USA).

用于蛋白质印迹的抗BamA多克隆抗体和pTrc99a-BamABCDE-CT8His质粒是Harris-Bernstein（美国国立卫生研究院）的礼物。用于蛋白质印迹的抗SurA多克隆抗体是Jean-François Collet（比利时UCLouvain）的慷慨礼物。BamA耗竭菌株（JCM166），SurA缺失菌株（AR208）和BamA互补载体（pZS21-BamA-NT6His）由Tom Silhavy（美国普林斯顿）慷慨提供。

All materials are available from the authors upon request.Author informationAuthor notesJim E. HornePresent address: Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1GA, UKRomany J. HornePresent address: Steinmetz Building, Granta Park, Great Ab.

。作者信息作者注释Jim E.HornePresent地址：剑桥网球场路生物化学系，CB2 1GA，UKRomany J.HornePresent地址：Granta Park Steinmetz Building，Great Ab。

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PubMed Google ScholarContributionsK.L.F. and J.E.H. contributed equally to this study and are designated as joint first authors. K.L.F. collected and processed all cryoEM data. J.E.H. led on construct design, molecular biology and protein production, assisted by R.J.H. Microbiology experiments were performed by KLF and JEH, as were proteomics experiments and data analysis (assisted by ANC).

PubMed谷歌学术贡献SK。五十、 F.和J.E.H.对这项研究做出了同样的贡献，并被指定为联合第一作者。K、 L.F.收集并处理了所有cryoEM数据。J、 E.H.在R.J.H.的协助下领导构建体设计，分子生物学和蛋白质生产。微生物学实验由KLF和JEH进行，蛋白质组学实验和数据分析（由ANC协助）也是如此。

J.A.C. performed single-molecule fluorescence experiments and data analysis. N.B. and T.F.S. produced and purified darobactin-B. The paper was written and edited by all authors, and all authors contributed to experiment design. The project was supervised by S.E.R. and N.A.R.Corresponding authorsCorrespondence to.

J、 A.C.进行了单分子荧光实验和数据分析。N、 B.和T.F.S.生产并纯化了darobactin-B。该论文由所有作者撰写和编辑，所有作者都为实验设计做出了贡献。该项目由S.E.R.和N.A.R.通讯作者通信监督。

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Reprints and permissionsAbout this articleCite this articleFenn, K.L., Horne, J.E., Crossley, J.A. et al. Outer membrane protein assembly mediated by BAM-SurA complexes.

转载和许可本文引用本文Fenn，K.L.，Horne，J.E.，Crossley，J.A。等人。BAM-SurA复合物介导的外膜蛋白组装。

Nat Commun 15, 7612 (2024). https://doi.org/10.1038/s41467-024-51358-xDownload citationReceived: 11 April 2024Accepted: 02 August 2024Published: 01 September 2024DOI: https://doi.org/10.1038/s41467-024-51358-xShare 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》157612（2024）。https://doi.org/10.1038/s41467-024-51358-xDownload引文接收日期：2024年4月11日接受日期：2024年8月2日发布日期：2024年9月1日OI：https://doi.org/10.1038/s41467-024-51358-xShare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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