Molecular Mechanisms of Mechanosensitive Channel Gating

 描述（由申请人提供）：研究大电导率的细菌机械敏感通道MscL的一个原因是，它已经并将继续作为研究机械感觉转导的分子范例。其晶体结构似乎是M的“近闭合”状态。结核病MscL，该通道通过允许研究人员将遗传和分子分析与电生理学结合在结构模型上，大大推进了该领域。因此，从E.大肠杆菌（Ec-MscL），这是第一个明确的机械敏感通道确定，继续作为一个易于处理的模型，用于确定蛋白质如何传感和响应膜张力的原则。然而，研究MscL的另一个新兴原因是该通道在维持微生物的渗透稳态方面起着至关重要的作用。它通常作为一个生物紧急释放阀;在渗透压下降时，它打开一个巨大的30 μ m孔，允许快速释放许多积累的细胞质成分，包括钾和谷氨酸，从而防止细胞溶解。当通道门控不当时，它可能导致微生物细胞死亡;因此它是潜在抗生素的可行药理学靶标。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通道的小分子探针。从一个高通量筛选（HTS）设施在校园里，我们已经确定了18个新的化合物，抑制E。大肠杆菌中的MscL依赖的方式;令人惊讶的是，另一个命中是链霉素，虽然不是其主要的作用机制，似乎直接结合并增加打开MscL通道的可能性;链霉素似乎也使用MscL作为进入细胞质的通道。在研究链霉素-MscL相互作用中，我们已经开发和改进了用于研究MscL-配体相互作用的测定。这些包括（1）在存在和不存在表达的Ec-MscL、MscL直向同源物或作为阴性对照的无关细菌机械敏感通道MscS的情况下测定化合物的最小抑制浓度（MIC），（2）测量化合物在体内诱导钾和谷氨酸从细胞流出的能力，（3）通过分子动力学模拟预测化合物结合位点及其如何修饰蛋白质，以及（4）通过生物化学和诱变手段测试这些预测。我们将使用这一系列的测定来确定在HTS中鉴定的另外两种非常有前途的化合物如何与MscL结合并调节其活性。这些研究将深入了解机械敏感通道门控机制;此外，MscL与一种或多种这些化合物的共结晶可能会产生MscL的开放状态结构，这些发现最终可能导致新一代抗生素。

 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.