Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate.
Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate.
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DOI：
10.3389/fchem.2023.1113885
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发表时间：
2023
影响因子：
5.5
通讯作者：
中科院分区：
文献类型：
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在人类病原体S.金黄色葡萄球菌受agr（辅助基因调节器）群体感应（QS）系统调节，该系统在多种革兰氏阳性细菌中是保守的。agr QS信号分子是通过跨膜肽酶AgrB对AgrD前肽的初始加工产生的自诱导肽（AIP）。由于缺乏AgrB和AgrBD相互作用的结构信息，我们使用同源建模和分子动力学（MD）退火来模拟AgrB和AgrD在模型膜和溶液中的构象。这些揭示了AgrB的六螺旋跨膜结构域（6 TMD）拓扑结构。在溶液中，AgrD表现为无序肽，在AgrB存在和不存在的情况下，其N-末端与膜结合。在计算机模拟中，AgrD和二聚AgrB的膜复合物显示分别负责初始结合和处理的非等效AgrB单体。通过利用金黄色葡萄球菌中的分裂荧光素酶测定，我们提供了实验证据，AgrB直接与自身和AgrD相互作用。我们证实了在体外形成的AgrBD复合物和AIP的生产后，使用从大肠杆菌表达AgrB或纯化的AgrB和T7标记的AgrD的膜蛋白质印迹。AgrB和AgrD形成稳定的复合物在洗涤剂胶束揭示使用同步辐射CD（SRCD）和朗道分析与增强的AgrD存在下的AgrB的热稳定性一致。通过小角X-射线散射从Proteodesulfant胶束中观察到AgrD提供后AgrB的构象改变。AgrB和AgrD的原子描述已经获得了与AgrB 6 TMD膜拓扑结构和AgrBD分子复合物在体外和体内的存在的确认一起。
Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo.
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影响因子：
14.8
作者：
Kozakov D;Hall DR;Xia B;Porter KA;Padhorny D;Yueh C;Beglov D;Vajda S
通讯作者：
Vajda S
影响因子：
6.1
作者：
Konarev, PV;Volkov, VV;Svergun, DI
通讯作者：
Svergun, DI
单个副本集成向量，该矢量在金黄色葡萄球菌染色体上的工程地点集成。
影响因子：
1.8
作者：
Lei MG;Cue D;Alba J;Junecko J;Graham JW;Lee CY
通讯作者：
Lee CY
影响因子：
5.2
作者：
Cheung GYC;Bae JS;Otto M
通讯作者：
Otto M
影响因子：
14.9
作者：
Drozdetskiy A;Cole C;Procter J;Barton GJ
通讯作者：
Barton GJ