毒素蛋白细胞膜自发成孔过程中分子内力驱动构象变化的机理研究

The ability to coordinate structural changes over long distances is a property of many (and perhaps all) proteins, from enzymes to ion channels to molecular motors. However, except for a few cases, the underlying physical mechanism of this structural coordination is not understood, a significant unresolved problem in protein biophysics. This is partly owing to the dynamic nature of the coordination, which is generally difficult to characterize by x-ray crystallography, as well as the subtlety of the structural changes that are often apparently involved. In contrast, many bacterial pore forming toxins undergo dramatic changes in conformation during their self-assembly into a pore, spontaneously converting from a water-soluble to a membrane-spanning conformation. With such large-scale structural changes in a self-assembled process, these fascinating toxins provide ideal model systems to identify paradigms of structural coordination in proteins. The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins secreted by Gram-positive pathogens that cause gas gangrene (that plagued many victims of the 2008 Sichuan earthquake), food poisoning, and pneumonia, among other diseases. They have also recently been found to share major features with the MACPF protein family that includes the human lysin, perforin. In this proposal, we will study the coordination of structural changes in the prototypical member of the CDC family, perfringolysin O (PFO), to establish general principles governing long-range structural coordination in this ubiquitous family of cytotoxins, which will likely apply to many other proteins, as well as provide a rational basis for future exploration of therapeutic targets and effective regions for re-engineering. In particular, our preliminary data indicate that the hydrophobic forces driving membrane insertion are transmitted across the protein to generate compressive forces on a distant region, which then catalyzes its change in conformation. Hence, the distant structural changes in PFO appear to be coordinated by intra-protein forces, a novel finding not previously described by others. Based on these findings, we propose a multifaceted approach, including extensive molecular dynamics simulations and several unique single molecule approaches, to provide the essential atomic-level structural and energetic details of this phenomenon and establish a solid mechanistic understanding of this process. Site-directed mutagenesis of PFO, as well as investigations of related members of the CDC family, pneumolysin and listeriolysin O, will also be undertaken to further establish this principle and its generality. Finally, we will explore specific modifications and small molecule inhibitors to the regions identified by this work to play a critical role in this mechanism for use in already promising biotechnological and clinical applications.

长距离结构协同变化普遍存在于酶、离子通道、分子马达等很多蛋白分子中。这种纳米尺度构型变化的精确物理学机制是当前生物物理领域中有待解决的重大问题之一。以perfringolysin O (PFO)为代表的细菌成孔毒素在插入质膜自组装成孔过程中结构变化明显，是探索这一问题的重要模型之一。根据前期工作，我们提出驱动蛋白插入膜中的疏水作用产生分子内作用力，并在分子内传递导致长距离外区域的结构变化，完成成孔过程。这一全新的机制与现有模型有本质的不同。本项目中，我们将使用多种单分子研究方法，从原子水平结构和能量变化来解析PFO在膜上成孔过程中的协同效应，证实并完善这一模型。我们还将通过PFO定点突变体及另两种相关蛋白，进一步探索这一模型的普适意义和适用范围。该模型的确立不但为大尺度构型变化提供了一个全新的工作机制，也为设计制备功能性成孔蛋白，寻找干预靶点等提供了重要基础。

本项目的总体目标为应用理论模拟与单分子技术手段研究细菌成孔毒素蛋白PFO的成孔机制。针对上述目标，本项目通过分子动力学模拟推测了PFO的成孔折叠过程，并通过原子力显微镜单分子力学方法等多种手段验证了该推测，证实了项目申请书中所提出的机制假设，揭示了PFO中与细胞膜作用的疏水区域产生足够内力导致PFO大尺度构型变化进行成孔折叠的物理机理。与此同时，通过原子力显微镜高分辨成像等方法，发现了PFO单体在自组装形成聚合物的过程中六聚体中间态的存在，从而提出了PFO自主装成孔的可能的物理模型。并通过气单胞菌溶素（aerolysin）的原子力显微镜高分辨成像，获得其在自然状态下不同于已有模型的星形结构。这些现象与机制的发现为设计针对PFO的功能药物提供了新的靶点，同时也为研究与PFO类似的成孔蛋白结构与成孔机理研究提供了方法学基础与模型指导。