ATP-dependent interactions of a cargo protein with the transmembrane domain of a polypeptide processing and secretion ABC transporter.
ATP-dependent interactions of a cargo protein with the transmembrane domain of a polypeptide processing and secretion ABC transporter.
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货物蛋白与多肽加工和分泌 ABC 转运蛋白的跨膜结构域之间的 ATP 依赖性相互作用。
DOI：
10.1074/jbc.ra120.014934
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Mchaourab,HassaneS
中科院分区：
文献类型：
--
作者：
Rahman,Suhaila;Mchaourab,HassaneS
由ATP结合和水解的能量提供动力，含有蛋白酶的ABC转运蛋白（PCAT）穿过膜疏水屏障输出两亲性和亲水性细菌素和群体感应蛋白。货物蛋白具有被半胱氨酸蛋白酶结构域（称为C39结构域或称为肽酶（PEP）结构域）切割的N-末端前导肽。PCAT和货物蛋白之间相互作用的序列和结构决定因素知之甚少，但这种相互作用是运输机制的一个核心方面。在这里，我们证明了ATP依赖的，平衡结合的货物蛋白的跨膜结构域（TMD）的PCAT后，由PEP结构域的前导肽的去除。货物蛋白与缺乏PEP结构域的PCAT 1变体的结合通过荧光或自旋标记的光谱特性的变化来检测。此外，我们发现类似的能量的结合，无论存在的前导肽，这表明，虽然PEP结构域用于识别和方向，与TMD的相互作用是亲和力的主要贡献者。这些发现与最近的一项研究直接矛盾，该研究声称TMD不与货物蛋白相互作用;而是充当穿过双层的“特氟隆样”导管（Kieuvongngam，V.，奥利纳雷斯警察局B.，Palillo，A.，奥尔德姆，M. L.，柴特，B。T.，和Chen，J.（2020）Structural basis of substrate recognition by a polypeptide processing and secretion transporter.eLife9，e51492）。从我们的数据中出现的运输模型的一个显着特征调用PCAT和它们的货物蛋白之间的稳定复合物后，加工的前导肽和之前的ATP依赖的交替访问，将货物蛋白易位到细胞外侧。
Powered by the energy of ATP binding and hydrolysis, protease-containing ABC transporters (PCATs) export amphipathic and hydrophilic bacteriocin and quorum-sensing proteins across the membrane hydrophobic barrier. The cargo proteins have N-terminal leader peptides that are cleaved off by the cysteine protease domain, referred to as the C39 domain, or referred to as the peptidase (PEP) domain. The sequence and structural determinants of the interaction between PCATs and cargo proteins are poorly understood, yet this interaction is a central aspect of the transport mechanism. Here, we demonstrate the ATP-dependent, equilibrium binding of the cargo protein to the transmembrane domain (TMD) of a PCAT subsequent to the removal of the leader peptide by the PEP domain. Binding of the cargo protein to PCAT1 variants devoid of the PEP domain is detected through changes in the spectroscopic properties of fluorescent or spin label. Moreover, we find similar energetics of binding regardless of the presence of the leader peptide, suggesting that although the PEP domain serves for recognition and orientation, interaction with the TMD is the main contributor to the affinity. These findings are in direct contradiction with a recent study claiming that the TMD does not interact with the cargo protein; rather acting as a “Teflon-like” conduit across the bilayer (Kieuvongngam, V., Olinares, P. D. B., Palillo, A., Oldham, M. L., Chait, B. T., and Chen, J. (2020) Structural basis of substrate recognition by a polypeptide processing and secretion transporter.eLife9, e51492). A distinctive feature of the transport model emerging from our data invokes a stable complex between PCATs and their cargo proteins following processing of the leader peptide and prior to ATP-dependent alternating access that translocates the cargo protein to the extracellular side.