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体，称为转位酶通道，在脂类双层之间移动。这些类型的转运蛋白对于膜包裹细胞器的形成和蛋白质分泌是至关重要的。转位酶通道也被病原菌用来将细胞毒蛋白和多肽输送到真核宿主细胞。然而，蛋白质易位的分子基础却知之甚少。已经有几个模型被用来描述化学或跨膜电位梯度能源如何被转化为促进折叠和易位的定向机械力。一方面，延伸链模型认为转运肽的转运通道是一个静态结构，它是一个几乎没有螺旋结构的延伸链。在此模型中， 肽是由质子驱动的布朗棘轮来解释的。另一方面，螺旋紧凑模型假设移位链从扩链构象收缩为螺旋构象。这种构象转变是通过通道中能够适应螺旋结构的变构构象变化来协调的。变构转变可以由位于转运体长度上的多肽夹活性部位的质子结合和动态转变来触发。以炭疽毒素为模型系统，利用高分辨电子显微镜(EM)、单通道电生理学和交联质谱(MS)对这两种模型进行了区分。炭疽芽孢杆菌分泌三蛋白毒素--炭疽毒素，它由保护性抗原(PA)、致死因子(LF)和水肿因子(EF)组成。PA是转位酶通道，负责将酶因子LF和EF送入宿主细胞质。PA首先与LF和EF共同组装形成寡聚毒素复合体，然后内吞。在内体膜内，PA插入并形成一个狭窄的水通道，LF和EF通过该通道展开和移位到达另一侧。已发现PA通道内的多肽钳位是动态活性部位，可结合和释放EF和LF的转移链。最近的高分辨电子显微镜结构揭示了中心苯丙氨酸钳(ϕ钳)位置的狭窄构型。然而，这种结构没有考虑到单通道电生理学和ϕ-钳环中假定接触残基的遗传协变所预期的钳子的另一种配置。这些构型将通过对通道的突变和pH依赖的研究来进行结构和热力学分析。为了深入了解通道内移位的LF的结构配置，将捕获构象锁定的衬底进行详细的结构分析。相关性：对蛋白质转位机制的洞察与开发中和毒素的新方法以及先进技术具有翻译相关性，这些新方法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输送到细胞的多功能载体。

 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.