Structural determinants of sensing and gating in MscS-like channels

机械敏感通道在渗透调节中起着重要作用，保护细菌免受低渗透休克。我们的研究集中在大肠杆菌中的MSCs样通道，它有六个类似物，促进对环境变化的分级反应。MSCs样通道有一个共同的七聚体结构，包括一个围绕着孔入口的有窗的胞液前庭和外围的膜锚定的桨，这是由每个亚单位贡献的。桨的大小根据副翼的不同而在2到10个螺旋之间变化。根据我们最近确定的中型YNAI和大型YbiO的结构，我们假设感测、门控和电导的结构决定因素编码在通道的模块化体系结构中。我们认为感测模块由球拍组成。压力响应是由桨上带正电的残留物与脂质的相互作用来调节的。门控可能编码在孔螺旋中疏水性封闭之前的一小段15个氨基酸中。YNAI通过缩短毛孔来关门，这与典型的小尺寸MSCs的机制明显不同。我们认为，毛孔缩短是在一个GGxGG基序中编码的，该基序专用于包括YNAI在内的一系列通道。最后，电导被编码在前庭和中央孔中，跟随着门控基序。我们认为这三个模块是可互换的单元，如果组合成嵌合通道，将显示出各自供体通道的功能特征。我们建议在YNAI背景下检验这些假设。因此，我们将产生突变体，询问桨中正电荷对脂质结合和压力反应的重要性。其他突变体将在YNAI背景下测试自然产生的富含甘氨酸的基序的门控特性，目的是确定哪些基序通过缩短毛孔来门，哪些保持在MSCs中观察到的毛孔长度。最后，我们将生成嵌合通道，其中YNAI模块将被来自其他类似MSCS的通道的相应模块替换，以证明这些模块是可互换的，并且仍然可以组装成功能通道。突变和嵌合体将通过测量它们保护细菌免受低渗透休克的能力来表征。膜片钳实验将量化压力、通道开放的阈值、开放通道的电导以及进一步的门控特性。通过电子冷冻显微镜和图像处理确定结构将识别门控机制中涉及的构象变化，并将功能变化映射到结构特征。综上所述，这将产生对MSCs样通道中结构功能关系的全面理解，从而阐明这一非常多样化的通道家族在自然界中的作用。

Mechanosensitive channels play an important role in osmoregulation, protecting bacteria against hypo-osmotic shock. Our research focusses on MscS-like channels in E.coli, which has six paralogues that promote a graded response to environmental changes. MscS-like channels have a common heptameric architecture comprising a fenestrated cytosolic vestibule that surrounds the entrance to the pore and membrane anchored paddles at the periphery which are contributed by each subunit. The size of the paddles varies between 2 and 10 helices depending on the paralogue. Based on our recently determined structures of the medium-sized YnaI and the large-sized YbiO, we hypothesize that the structural determinants for sensing, gating and conductance are encoded in the modular architecture of the channels. We think that the sensing module comprises the paddles. The pressure response is modulated by the interaction of positively charged residues in the paddles with lipids. Gating is probably encoded in a short stretch of 15 amino acids in the pore helices preceding the hydrophobic seal of the pore. YnaI gates by shortening the pore, which is markedly different to the mechanism of the prototypic small-sized MscS. We think that pore-shortening is encoded in a GGxGG motif specific to a subset of channels including YnaI. Finally, the conductance is encoded in the vestibule and the central pore following the gating motif. We suggest that the three modules are interchangeable units and if combined into chimeric channels will exhibit the functional characteristics of the respective donor channels. We propose to test these hypotheses in an YnaI background. Therefore, we will generate mutants that interrogate the importance of the positive charges in the paddles for lipid binding and pressure response. Other mutants will test the gating characteristics of naturally occurring Gly-rich motifs in an YnaI background with the aim to establish which motifs gate by pore shortening and which maintain the pore length as observed in MscS. Finally, we will generate chimeric channels, in which YnaI modules will be replaced with the respective modules from other MscS-like channels to proof that the modules are interchangeable and still assemble into functional channels. Mutations and chimeras will be characterized by measuring their ability to protect bacteria against hypo-osmotic shock. Patch-clamp experiments will quantify the pressure, threshold for channel opening, the conductance of the open channels and further gating characteristics. Structure determination by electron cryo microscopy and image processing will identify the conformational changes involved in the gating mechanism and will map functional changes to structural characteristics. Together this will generate a comprehensive understanding of the structure function relationship in MscS-like channels that will shed light on the role of this very diverse channel family in nature.