Structural determinants of sensing and gating in MscS-like channels

MscS 类通道中传感和门控的结构决定因素

• 批准号: 343886090
• 负责人: Professorin Dr. Bettina Böttcher
• 金额: --
• 依托单位: Lehrstuhl für Biochemie I
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/343886090?language=en
• 关键词: Structural; determinants; sensing; gating; MscS

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.

机械敏感性通道在渗透调节中起重要作用，保护细菌免受低渗休克。我们的研究重点是大肠杆菌中的MSC样通道，它有六个旁系同源物，促进对环境变化的分级反应。 MSC样通道具有共同的七聚体结构，包括围绕孔入口的有孔胞质前庭和由每个亚基贡献的外周处的膜锚定桨。桨的大小在2和10个螺旋之间变化，这取决于桨的大小。基于我们最近确定的中型YnaI和大型YbiO的结构，我们假设传感，门控和电导的结构决定因素编码在通道的模块化结构中。我们认为传感模块包括桨。压力响应是由桨状结构中带正电荷的残基与脂质的相互作用来调制的。门控可能编码在孔螺旋中的疏水密封之前的15个氨基酸的短片段中。YnaI通过缩短孔进行门控，这与原型小尺寸MscS的机制明显不同。我们认为，孔缩短编码在一个GGxGG基序特定的一个子集的通道，包括YnaI。 最后，在前庭和中央孔中编码传导性，遵循门控基序。我们认为，这三个模块是可互换的单位，如果结合到嵌合通道将表现出各自的供体通道的功能特性。我们建议在YnaI背景下测试这些假设。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背景下测试天然存在的富含Gly基序的门控特征，目的是确定哪些基序通过孔缩短进行门控，哪些基序保持MscS中观察到的孔长度。最后，我们将生成嵌合通道，其中YnaI模块将被其他类似MSCS通道的相应模块替换，以证明模块是可互换的，并且仍然组装成功能通道。突变和嵌合体将通过测量其保护细菌免受低渗休克的能力来表征。膜片钳实验将量化压力、通道开放的阈值、开放通道的电导和进一步的门控特征。通过电子冷冻显微镜和图像处理进行的结构测定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。总之，这将产生一个全面的理解的结构功能关系的MSCS样渠道，将揭示这个非常多样化的渠道家庭的作用的性质。