Molecular analyses of toxin nanopore structural dynamics

 描述（由申请人提供）：蛋白质通过膜嵌入的转运蛋白（称为转位酶通道）穿过脂质双层。这些类型的转运蛋白对于膜封装细胞器的形成和蛋白质分泌至关重要。转位酶通道也被病原菌用来将细胞毒性蛋白和肽递送到真核宿主细胞中。然而，人们对蛋白质易位的分子基础知之甚少。已调用多个模型来描述如何将化学或跨膜势梯度能源转换为促进展开和易位的定向机械力。一方面，延长链模型认为通道是静态结构，转位肽通过该静态结构转位为几乎没有螺旋结构的延长链。在此模型中，净运动 肽是通过质子驱动的布朗棘轮来解释的。另一方面，螺旋压缩模型假设易位链从延长链构象收缩为螺旋构象。这种构象转变是通过可容纳螺旋结构的通道中的变构构象变化来协调的。变构转变可由质子结合和沿转运蛋白长度定位的多肽夹活性位点的动态转变触发。该提案以炭疽毒素作为模型系统，试图利用高分辨率电子显微镜（EM）、单通道电生理学和交联质谱（MS）来区分这两种模型。炭疽杆菌分泌三蛋白毒素——炭疽毒素，由保护性抗原（PA）、致死因子（LF）和水肿因子（EF）组成。 PA 是将酶因子 LF 和 EF 传递到宿主细胞质中的转位酶通道。 PA 首先与 LF 和 EF 共同组装，形成被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 通过该通道展开并易位到达另一侧。 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的易位链。最近的高分辨率电子显微镜结构揭示了中央苯丙氨酸夹（夹）位点的狭窄构型。然而，这种结构并没有考虑从单通道电生理学和 phi-clamp 环中假定的接触残基的遗传共变中预期的钳位的替代配置。这些构型将通过通道的突变和 pH 依赖性研究进行结构和热力学分析。为了深入了解通道内易位 LF 的结构配置，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中和毒素新方法的开发以及先进技术具有转化相关性，这些技术利用毒素作为纳米孔生物传感器和将异源抗原和细胞毒素送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.