Molecular analyses of toxin nanopore structural dynamics

 描述（由申请人提供）：蛋白质通过膜包埋转运蛋白（称为转位酶通道）跨脂质双层移动。这些类型的转运蛋白对膜包封细胞器的形成和蛋白质分泌至关重要。易位酶通道也被病原菌用于将细胞毒性蛋白质和肽递送到真核宿主细胞中。然而，蛋白质易位的分子基础知之甚少。已经调用了几个模型来描述化学或跨膜电位梯度能量源如何可以被转换成促进解折叠和易位的定向机械力。一方面，延伸链模型认为通道是一个静态结构，通过该通道，易位肽作为具有很少螺旋结构的延伸链易位。在这个模型中， 肽是由质子动力的布朗棘轮解释。另一方面，螺旋压缩模型假设易位链从伸展链构象收缩为螺旋构象。这种构象转变是由通道中的变构构象变化协调的，该变构构象变化可以容纳螺旋结构。变构转换可以由质子结合和位于沿着转运蛋白长度的多肽钳活性位点中的动态转换触发。以炭疽毒素为模型系统，本研究试图利用高分辨电子显微镜（EM）、单通道电生理学和交联质谱（MS）来区分这两种模型。炭疽杆菌分泌由保护性抗原（PA）、致死因子（LF）和水肿因子（EF）组成的三蛋白毒素-炭疽毒素。PA是将酶因子LF和EF递送到宿主细胞质中的移位酶通道。PA首先与LF和EF共组装以形成内吞的寡聚毒素复合物。在内体膜内，PA插入并形成狭窄的水通道，LF和EF通过该水通道展开并移位通过该水通道到达另一侧。PA通道内的多肽钳位点被发现是动态活性位点，可以结合和释放EF和LF的转运链。最近的高分辨率电子显微镜结构揭示了中央苯丙氨酸钳（Phenylalanine clamp）位点的狭窄构型。然而，这种结构并不能解释从单通道电生理学和假定的接触残基的遗传共变异中预期的钳环的替代构型。这些配置将通过对通道的突变和pH依赖性研究进行结构和化学分析。为了深入了解通道内移位LF的结构构型，将捕获构象锁定的底物以进行详细的结构分析。相关性：对蛋白质易位机制的深入了解与开发中和毒素的新方法以及推进技术具有翻译相关性，这些技术利用毒素作为纳米孔生物传感器和用于异源抗原和细胞毒素进入细胞的多功能递送载体。

 DESCRIPTION (provided by applicant): Proteins move across lipid bilayers through membrane-embedded transporters, called translocase channels. These types of transporters are critical to the formation of membrane-encapsulated organelles and protein secretion. Translocase channels are also used by pathogenic bacteria to deliver cytotoxic proteins and peptides into eukaryotic host cells. The molecular basis of protein translocation, however, is poorly understood. Several models have been invoked to describe how a chemical or transmembrane potential gradient energy source can be transduced into a directed mechanical force that promotes unfolding and translocation. On one hand, an extended-chain model considers the channel to be a static structure through which the translocating peptide translocates as an extended chain with little helical structure. In this model, net movement of the peptide is explained by a proton-powered Brownian ratchet. On the other hand, the helix-compaction model hypothesizes that the translocating chain contracts from an extended-chain conformation to a helical one. This conformational transition is coordinated by an allosteric conformational change in the channel that can accommodate helical structure. The allosteric transition may be triggered by proton binding and dynamic transitions in the polypeptide clamp active sites located along the length of the transporter. Using anthrax toxin as model system, this proposal seeks to distinguish these two models using high-resolution electron microscopy (EM), single-channel electrophysiology, and cross-linking mass spectrometry (MS). The bacterium, Bacillus anthracis secretes the three-protein toxin, anthrax toxin, which is composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA is the translocase channel that delivers the enzymatic factors, LF and EF, into the host cytosol. PA first co-assembles with LF and EF to form an oligomeric toxin complex that is endocytosed. Within the endosomal membrane, PA inserts and forms a narrow aqueous passageway through which LF and EF unfold and translocate through to reach the other side. Polypeptide clamp sites within the PA channel have been found to be dynamic active sites, which can bind and release the translocating chain of EF and LF. A recent high- resolution electron microscopy structure has revealed a narrow configuration of the central phenylalanine clamp (ϕ clamp) site. However, this structure does not account for an alternate configuration of the clamp anticipated from single-channel electrophysiology and genetic co-variation of putative contacting residues in the ϕ-clamp loop. These configurations will be analyzed structurally and thermodynamically by mutational and pH-dependent studies of the channel. To gain insight on the structural configurations of the translocating LF inside the channel, conformationally locked substrates will be trapped for detailed structural analysis. Relevance: Insight on the mechanism of protein translocation is of translational relevance to the development of novel methods to neutralize the toxin and also to advancing technologies, which exploit toxins as nanopore biosensors and versatile delivery vehicles for heterologous antigens and cytotoxins into cells.