Molecular Mechanisms of Mechanosensitive Channel Gating

 描述(申请人提供)：研究细菌大电导机械敏感通道MSCL的一个原因是它已经并将继续作为机械感觉转导研究的分子范例。该通道的晶体结构看起来像是结核分枝杆菌的MSCL，它使研究人员能够将遗传和分子分析与电生理学结合在一个结构模型上，从而极大地推动了该领域的发展。因此，来自大肠杆菌的MSCL(EC-MSCL)是第一个被发现的明确的机械敏感通道，它继续作为一个容易处理的模型来确定蛋白质如何感知和响应膜张力的原理。然而，研究MSCL的另一个新出现的原因是，该通道在维持微生物的渗透平衡方面起着至关重要的作用。它通常用作生物紧急释放阀；在渗透性下激波时，它会打开一个巨大的30？孔，允许许多积累的细胞质成分快速释放，包括钾和谷氨酸，从而防止细胞溶解。当通道不适当地关闭时，可能会导致微生物细胞死亡；因此，它是潜在抗生素的可行药理靶点。从历史上看，MSCL功能和药理学研究的局限性之一是完全缺乏结合和调节该通道的小分子探针。在校园的高通量筛选(HTS)设施中，我们已经鉴定出18种新的化合物，它们以依赖于MSCL的方式抑制大肠杆菌的生长；令人惊讶的是，另一种打击是链霉素，尽管它不是其主要作用机制，但似乎直接与MSCL结合并增加打开MSCL通道的可能性；链霉素似乎也利用MSCL作为进入细胞质的通道。在链霉素-MSCL相互作用的研究中，我们发展和改进了研究MSCL-配体相互作用的方法。这些步骤包括(1)确定化合物在存在和不存在表达的EC-MSCL、MSCL同源物或无关的细菌机械敏感通道MSCs的情况下的最低抑菌浓度(MIC)，作为阴性对照；(2)测量化合物在体内诱导钾和谷氨酸从细胞中流动的能力；(3)通过分子动力学模拟预测化合物的结合位置以及它如何修饰蛋白质；以及(4)通过生化和突变方法检验这些预测。我们将使用这一系列的分析来确定HTS中确定的另外两种非常有希望的化合物如何与MSCL结合并调节其活性。这些研究将深入了解机械敏感的通道门控机制；此外，MSCL与这些化合物中的一种或多种共结晶可能会产生MSCL的开放状态结构，这些发现最终可能导致新一代抗生素的出现。

 DESCRIPTION (provided by applicant): One reason to study the bacterial mechanosensitive channel of large conductance, MscL, is that it has, and will continue to serve as a molecular paradigm for the investigation of mechanosensory transduction. With a crystal structure of what appears to be a 'nearly-closed' state of M. tuberculosis MscL, the channel has advanced the field considerably by allowing researchers to overlay genetic and molecular analyses, coupled with electrophysiology, onto a structural model. Thus, MscL from E. coli (Ec-MscL), which was the first definitive mechanosensitive channel identified, continues to serve as a tractable model for determining principles for how a protein senses and responds to membrane tension. However, another emerging reason to study MscL is that the channel serves a vital function in maintaining osmotic homeostasis of microbes. It normally serves as a biological emergency release valve; upon osmotic downshock it opens a huge 30Å pore that allows for the rapid release of many accumulated cytoplasmic components, including potassium and glutamate, thus preventing cell lysis. When the channel gates inappropriately it can lead to the death of the microbial cell; it thus is a viable pharmacological target for potential antibiotics. Historically,one of the limitations in the study of MscL function and pharmacology has been the total lack of small molecular probes that bind and modulate the channel. From a High Throughput Screening (HTS) facility on campus, we have identified 18 novel chemical compounds that inhibit the growth of E. coli in a MscL-dependent manner; surprisingly, an additional hit was streptomycin, which, although not its primary mechanism of action, appears to directly bind to and increase the probability of opening the MscL channel; streptomycin also appears to use MscL as a passageway into the cells cytoplasm. In studying streptomycin-MscL interactions, we have developed and refined assays for studying MscL-ligand interactions. These include (1) determining the minimal inhibitory concentrations (MIC) of compounds in the presence and absence of expressed Ec-MscL, MscL orthologues, or the unrelated bacterial mechanosensitive channel MscS as a negative control, (2) measuring the ability of a compound to induce fluxes of potassium and glutamate from the cell in vivo, (3) predicting the compound binding site and how it modifies the protein by using molecular dynamic simulations, and (4) testing these predictions by biochemical and mutagenic means. We will use this array of assays to determine how two additional and very promising compounds identified in the HTS bind to MscL and modulate its activity. These studies will yield insight into mechanosensitive channel gating mechanisms; in addition, co-crystallization of MscL with one or more of these compounds may yield an open state structure for MscL, and the findings could eventually lead to a new generation of antibiotics.