AbstractProtease-containing ABC transporters (PCATs) couple the energy of ATP hydrolysis to the processing and export of diverse cargo proteins across cell membranes to mediate antimicrobial resistance and quorum sensing. Here, we combine biochemical analysis, single particle cryoEM, and DEER spectroscopy in lipid bilayers along with computational analysis to illuminate the structural and energetic underpinnings of coupled cargo protein export.

摘要含蛋白酶的ABC转运蛋白（PCAT）将ATP水解的能量耦合到跨细胞膜加工和输出多种货物蛋白，以介导抗微生物抗性和群体感应。在这里，我们将脂质双层中的生化分析，单粒子cryoEM和DEER光谱与计算分析相结合，以阐明耦合货物蛋白质输出的结构和能量基础。

Our integrated investigation uncovers competitive interplay between nucleotides and cargo protein binding that ensures the latter’s orderly processing and subsequent transport. The energetics of cryoEM structures in lipid bilayers are congruent with the inferred mechanism from ATP turnover analysis and reveal a snapshot of a high-energy outward-facing conformation that provides an exit pathway into the lipid bilayer and/or the extracellular side.

我们的综合研究揭示了核苷酸和货物蛋白结合之间的竞争性相互作用，确保了后者的有序加工和随后的运输。脂质双层中cryoEM结构的能量学与ATP周转分析推断的机制一致，并揭示了高能外向构象的快照，该构象提供了进入脂质双层和/或细胞外侧的出口途径。

DEER investigation of the core ABC transporter suggests evolutionary tuning of the energetic landscape to fulfill the function of substrate processing prior to export..

DEER对核心ABC转运蛋白的研究表明，能量景观的进化调整可以在出口之前实现底物加工的功能。。

IntroductionABC transporters are exquisite molecular machines that traffic solutes across lipid bilayers in both directions powered by the energy of ATP hydrolysis1,2. Broadly classified into importers and exporters, functional specialization among ABC transporters led to a fascinating spectrum of molecular architectures3.

简介ABC转运蛋白是一种精致的分子机器，由ATP水解能量驱动，在脂质双层的两个方向上运输溶质1,2。ABC转运蛋白广泛分为进口商和出口商，功能专业化导致了一系列迷人的分子结构3。

A subfamily of ABC exporters specializes in the processing and translocation of entire protein domains across cell membranes. Referred to as Peptidase-Containing ABC transporters (PCATs), their architecture consists of a core homodimeric ABC exporter covalently linked to a cysteine protease domain (C39 or PEP domain)4,5,6,7.

ABC出口商的一个亚家族专门从事整个蛋白质结构域在细胞膜上的加工和转运。被称为含肽酶的ABC转运蛋白（PCAT），其结构由与半胱氨酸蛋白酶结构域（C39或PEP结构域）共价连接的核心同型二聚体ABC出口物组成4,5,6,7。

Cargos of PCATs includes bacteriocins and quorum-sensing polypeptides, fundamental bacterial defense strategies4,5,8,9.One of the most structurally characterized PCATs is PCAT1 from the thermophilic organism C. thermocellum10,11,12. The homodimeric core ABC transporter of PCAT1 has the canonical structure of ABC Type IV exporters with two cytoplasmic nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs).

PCAT的货物包括细菌素和群体感应多肽，基本的细菌防御策略4,5,8,9。最具结构特征的PCAT之一是来自嗜热生物体C.thermocellum10,11,12的PCAT1。PCAT1的同型二聚体核心ABC转运蛋白具有ABC IV型出口商的规范结构，具有两个细胞质核苷酸结合结构域（NBD）和两个跨膜结构域（TMD）。

PCAT1 cargo protein, hereafter referred to as Sub, was identified as a 90-residue polypeptide containing an N-terminal leader sequence, which is cleaved by the PEP domain of PCAT111. Initial crystal structures of PCAT1 in two conformations11 were followed by several cryoEM snapshots under different biochemical conditions10,12.

PCAT1货物蛋白（以下称为Sub）被鉴定为含有N端前导序列的90个残基的多肽，其被PCAT111的PEP结构域切割。PCAT1在两种构象11中的初始晶体结构之后是在不同生化条件下的几个cryoEM快照10,12。

Collectively the structural record revealed the molecular architecture of PCAT1, identified elements of its ATP-dependent conformational changes, and elucidated a partial view of its interactions with Sub.Persistently enigmatic, not only for PCAT1 but other PCATs as well13, has been the coupling mechanism of cargo protein export to the cycle of ATP turnover.

总的来说，结构记录揭示了PCAT1的分子结构，确定了其ATP依赖性构象变化的元素，并阐明了其与亚基相互作用的部分观点。不仅对于PCAT1，而且对于其他PCAT，持续神秘13，一直是货物蛋白质输出到ATP周转周期的耦合机制。

CryoEM structures of Sub-bound PC.

亚束缚PC的CryoEM结构。

(1)

(1)

where \({\theta }_{n}\) is the angle formed by \({C}_{\alpha }^{\left(n\right)}-{C}_{\alpha }^{\left(n+1\right)}-{C}_{\alpha }^{\left(n+2\right)}\) and \({d}_{n}\) is distance between \({O}^{(n)}\) and \({N}^{(n+4)}.\) \(f(\theta )\) and \(g(d)\) are score functions (both ranging from 0 to 1) quantifying the likelihood of \(\theta\) and \({d}\) being associated with an \(\alpha\)-helix.

其中\（{\ theta}\{n}\）是由\({C}_{\ alpha}^{\左（n \右）}-{C}_{\ alpha}^{\左（n+1 \右）}-{C}_{\ alpha}^{\左（n+2 \右）}\）和\({d}_{n} \）是\（{O}^{（n）}\）和\（{n}^{（n+4）}\）之间的距离\（f（\ theta）\）和\（g（d）\）是分数函数（范围从0到1），量化\（\ theta \）和\（{d}\）与\（\ alpha \）螺旋关联的可能性。

More specifically:$$f\left(\theta \right)=\frac{1-{\left(\frac{\theta -{\theta }_{0}}{\delta \theta }\right)}^{2}}{1-{\left(\frac{\theta -{\theta }_{0}}{\delta \theta }\right)}^{4}},\,g\left(d\right)=\frac{1-{\left(\frac{d}{{d}_{0}}\right)}^{6}}{1-{\left(\frac{d}{{d}_{0}}\right)}^{8}},$$.

更具体地说：$$f \左（\θ\右）=\ frac{1-{\左（\ frac{\θ-{\θ}}u0}{\δ\θ}\右）}^{2}{1-{\左（\ frac{\θ-{\θ}u0}{\δ\θ}\右）}^{4}，\，g \左（d \右）=\ frac{1-{\左（\ frac{d}{{d}_{0}}\右）}^{6}}{1-{\左（\frac{d}）{{d}_{0}}\右）}^{8}}，$$。

(2)

(2)

where \({\theta }_{0}\) and \(\delta \theta\) are 88 and 15 degrees, respectively and \({d}_{0}\) is 3.3 \(\mathring{\rm A}\). For the well-tempered metadynamics, the height and width of the gaussians were 1 kcal/mol and 0.05, respectively, and the pseudo temperature was 1200 K.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article..

其中\（{\θ}\{0}\）和\（\δ\θ\）分别为88度和15度\({d}_{0}\）是3.3 \（\mathring{\rm A}\）。对于温和的元动力学，高斯的高度和宽度分别为1千卡/摩尔和0.05，伪温度为1200 K。报告摘要有关研究设计的更多信息，请参阅与本文相关的Nature Portfolio Reporting Summary。。

Data availability

数据可用性

The data that support this study are available from the corresponding authors upon request. All cryoEM 3D density maps of the PCAT1 in nanodiscs in Fig. 2, Fig. 5, supplementary table 2 and supplementary table 6 have been deposited in the Electron Microscopy Data Bank under accession codes EMD-43405 (PCAT1_OC_ADP); EMD-43404 (PCAT1_IF_ADP/Sub); EMD-43406 (PCAT1_IF_ADP/Mg2+_Class2); EMD-43400 (PCAT1_IF_ADP/Sub/Mg2+); EMD-43401 (PCAT1*_IF_ADP/Sub); EMD-43402 (PCAT1_IF_Mg2+_Class2); EMD-43403 (PCAT1_OC_ATP/Sub); EMD-43394 (PCAT1_CC_ADP/Mg2+); EMD-43396 (PCAT1_CC_ADP/Mg2+/Sub); EMD-43393 (PCAT1_CC_ATP/Mg2+/Vi); EMD-43398 (PCAT1_CC_ATP); EMD-44015 (PCAT1_IF_ADP/ Mg2+_Class1); EMD-44402 (PCAT1_IF_ Mg2+_Class1)(supplementary table 2 and supplementary table 6).

支持这项研究的数据可应要求从通讯作者那里获得。图2，图5，补充表2和补充表6中纳米盘中PCAT1的所有cryoEM 3D密度图已以登录号EMD-43405（PCAT1\u OC\u ADP）保存在电子显微镜数据库中；EMD-43404（PCAT1\U IF\U ADP/Sub）；；EMD-43400（PCAT1\u IF\u ADP/Sub/Mg2+）；EMD-43401（PCAT1*\u IF\u ADP/Sub）；EMD-43402（PCAT1\U IF\U Mg2+\U Class2）；EMD-43403（PCAT1\u OC\u ATP/Sub）；EMD-43394（PCAT1\u CC\u ADP/Mg2+）；EMD-43396（PCAT1\u CC\u ADP/Mg2+/Sub）；EMD-43393（PCAT1\u CC\u ATP/Mg2+/Vi）；EMD-43398（PCAT1\u CC\u ATP）；EMD-44015（PCAT1\U IF\U ADP/Mg2+\U类别）；EMD-44402（PCAT1\U IF\UMg2+\U类别）（补充表2和补充表6）。

Atomic coordinates for the atomic models have been deposited in the Protein Data Bank under accession codes 8VPA (PCAT1_OC_ADP); 8VP9 (PCAT1_IF_ADP/Sub); 8VPB (PCAT1_IF_ADP/Mg2+_Class2); 8VP3 (PCAT1_IF_ADP/Sub/Mg2+); 8VP5 (PCAT1*_IF_ADP/Sub); 8VP6 (PCAT1_IF_Mg2+_Class2); 8VP8 (PCAT1_OC_ATP/Sub); 8VOX (PCAT1_CC_ADP/Mg2+); 8VOZ (PCAT1_CC_ADP/Mg2+/Sub); 8VOW (PCAT1_CC_ATP/Mg2+/Vi); 8VP1 (PCAT1_CC_ATP); 9AZL (PCAT1_IF_ADP/Mg2+_Class1); 9BAA (PCAT1_IF_ Mg2+_Class1).

原子模型的原子坐标已保存在蛋白质数据库中，登录号为8VPA（PCAT1\u OC\u ADP）；8VP9（PCAT1\u IF\u ADP/Sub）；8VPB（PCAT1\u IF\u ADP/Mg2+\u Class2）；8VP3（PCAT1\u IF\u ADP/Sub/Mg2+）；8VP5（PCAT1*\u IF\u ADP/Sub）；8VP6（PCAT1\u IF\u Mg2+\u Class2）；8VP8（PCAT1\u OC\u ATP/Sub）；8VOX（PCAT1\u CC\u ADP/Mg2+）；；8VOW（PCAT1\u CC\u ATP/Mg2+/Vi）；8VP1（PCAT1\u CC\u ATP）；9AZL（PCAT1\u IF\u ADP/Mg2+\u类别）；9BAA（PCAT1\U IF\UMg2+\U类别）。

The MD simulation data necessary to reproduce the data including equilibration and FEP simulation input files are deposited to the Zenodo repository [https://doi.org/10.5281/zenodo.13864175]. Previous published structures referred in the manuscript is 7T54. A source data file is available. Source data are provided with this paper..

重现数据所需的MD模拟数据（包括平衡和FEP模拟输入文件）保存在Zenodo存储库中[https://doi.org/10.5281/zenodo.13864175]。手稿中提到的先前发布的结构是7T54。源数据文件可用。本文提供了源数据。。

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Download referencesAcknowledgementsThis work was supported by NIH grant R35 GM152382 to Hassane S. Mchaourab and NIH grant R35GM147423 to Mahmoud Moradi. EM data collections were conducted at the Center for Structural Biology Cryo-EM Facility at Vanderbilt University. We acknowledge the use of the Glacios cryo-TEM, which was acquired by NIH grant S10 OD030292-01.

下载参考文献致谢这项工作得到了美国国立卫生研究院授予Hassane S.Mchaourab的R35 GM152382和授予Mahmoud Moradi的R35GM147423的支持。EM数据收集是在范德比尔特大学结构生物学冷冻EM设施中心进行的。我们承认使用了由NIH grant S10 OD030292-01获得的Glacios cryo-TEM。

The simulations were performed on Frontera at the Texas Advanced Computing Center (TACC) through LRAC award CHE21003, made possible by the National Science Foundation award OAC-1818253.Author informationAuthors and AffiliationsDepartment of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN, USARuojing Zhang, Kevin L.

这些模拟是通过LRAC奖CHE21003在德克萨斯州高级计算中心（TACC）的Frontera上进行的，该奖由国家科学基金会奖OAC-1818253授予。作者信息作者和附属机构范德比尔特大学分子生理学和生物物理学系，田纳西州纳什维尔，USARuojing Zhang，Kevin L。

Jagessar, Richard A. Stein, Erkan Karakas & Hassane S. MchaourabDepartment of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USAMatthew Brownd, Adithya Polasa & Mahmoud MoradiAuthorsRuojing ZhangView author publicationsYou can also search for this author in.

Jagessar，Richard A.Stein，Erkan Karakas＆Hassane S.MchaourabDepartment of Chemistry and Biochemistry，阿肯色大学，费耶特维尔，阿肯色州，美国马修·布朗德，Adithya Polasa＆Mahmoud MoradiAuthorsRuojing ZhangView作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsR.Z. performed the cryoEM experiments and the ATPase assays. K.J. performed DEER measurement in collaborations with R.S. A.P. and M.B. performed the MD simulation under the supervision of M.M. E.K. provided advice for structure determination and analysis.

PubMed谷歌学术贡献。Z、 进行了cryoEM实验和ATPase测定。K、 J.与R.S.A.P.和M.B.合作进行了DEER测量。在M.M.E.K.的监督下进行了MD模拟。为结构确定和分析提供了建议。

H.M. designed the research. R.Z., R.S. and H.M. analyzed, and interpreted the structures. H.M. wrote the manuscript with input from all authors.Corresponding authorCorrespondence to.

H、 M.设计了这项研究。R、 Z.，R.S.和H.M.分析并解释了这些结构。H、 M.在所有作者的意见下撰写了手稿。对应作者对应。

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Reprints and permissionsAbout this articleCite this articleZhang, R., Jagessar, K.L., Brownd, M. et al. Conformational cycle of a protease-containing ABC transporter in lipid nanodiscs reveals the mechanism of cargo-protein coupling.

转载和许可本文引用本文Zhang，R.，Jagessar，K.L.，Brownd，M。等人。脂质纳米盘中含有蛋白酶的ABC转运蛋白的构象循环揭示了货物-蛋白质偶联的机制。

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