Structural Studies of Lymphostatin

肠致病性E.大肠杆菌（EPEC）和肠出血性大肠杆菌（E.大肠杆菌（EHEC）变异体引起肠道和全身性疾病。EPEC是婴儿腹泻的主要原因，并在人与人之间直接传播。相比之下，人类的肠出血性大肠杆菌感染经常是由于接触反刍动物粪便引起的，通常涉及出血性腹泻和危及生命的肾脏并发症。淋巴抑制素是EPEC和大多数EHEC菌株共有的关键毒力因子之一，是目前在大肠杆菌中发现的最大的蛋白。杆菌抑癌素似乎是一种多功能的多结构域蛋白，既能抑制淋巴细胞的功能，又能促进细菌附着于宿主细胞。其中一个结构域是蛋白质N-末端一半的糖基转移酶（GT）结构域。类似的GT结构域也存在于其他细菌毒素如小球藻毒素中。保守的GT结构域仅占淋巴抑制素的一小部分，而淋巴抑制素的C-末端部分与小球藻毒素没有同源性。我们认为，淋巴抑制素激活和失活某些功能的构象转换和自蛋白水解在不同阶段的infection.Here，我们提出了测试这一假设的结构测定淋巴抑制素及其N-和C-末端片段，结合和不结合底物的电子冷冻显微镜和图像处理。这些研究将显示GT结构域位于全长蛋白质中的位置，以及GT结构域的淋巴抑制素特异性延伸是否位于修饰底物结合位点或不同结构域之间结构通讯的战略位置。 片段和全长lymphostatin之间的结构比较将启发，如果掩蔽和解蔽的关键网站是潜在的机制的功能转换auto-proteolysis.有证据表明，lymphostatin是本地化的细菌膜，但它是如何影响这些膜。为了解决这个问题，我们将重组淋巴抑素脂质体和研究蛋白脂质体的冷冻电子断层扫描和subtomography平均。这些研究将显示淋巴抑制素是否简单地装饰膜，从而形成其他因子的潜在结合平台，或者它是否调节膜，例如通过形成孔或病变。 总之，我们提出的调查将揭示淋巴抑制素的整体结构及其功能模块之间的相互作用。我们期望淋巴抑制素通过简单的结构变化和自身蛋白水解来激活或灭活某些功能，使其成为一种多用途工具。对这些机制的详细结构知识将是必要的，以干扰淋巴抑制素的作用，从而减少在未来的EHEC和EPEC感染的破坏性影响。

Enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) variants cause intestinal and systemic diseases. EPEC are a leading cause of infant diarrhoea and transmit directly between humans. By contrast EHEC infections in humans frequently arise from contact with ruminant faeces and often involve bloody diarrhoea and life-threatening renal complications. One of the key virulence factors common to EPEC and most EHEC strains is lymphostatin, which is the largest protein yet identified in E. coli. Lymphostatin appears to be a multifunctional, multidomain protein serving both, to inhibit the function of lymphocytes and to promote bacterial attachment to host cells. One of the domains is a glycosyltransferase (GT) domain in the N-terminal half of the protein. Similar GT-domains are also found in other bacterial toxins such as the Chlostridial toxins. The conserved GT-domain accounts for only a small fraction of lymphostatin, while the C-terminal part of lymphostatin shows no homology to the Chlostridial toxins. We think that lymphostatin activates and deactivates certain functionalities by conformational switching and by auto-proteolysis during different stages of infection.Here we propose to test this hypothesis by structure determination of lymphostatin and its N- and C-terminal fragments with and without bound substrate by electron cryo microscopy and image processing. These studies will show where the GT-domain is located in the full-length protein and whether the lymphostatin-specific extensions of the GT-domain are at strategic positions to either modify the substrate binding site or alternatively the structural communication between different domains. Structure comparison between fragments and full-length lymphostatin will enlighten if masking and unmasking of key sites are potential mechanisms of functional switching by auto-proteolysis.There is evidence that lymphostatin is localized at the bacterial membranes but it is unknown how it affects these membranes. To address this question, we will reconstitute lymphostatin into liposomes and study the proteoliposomes by cryo electron tomography and subtomogram averaging. These investigations will show whether lymphostatin simply decorates the membranes and thus forms a potential binding platform for other factors or whether it modulates the membranes, e.g. by forming pores or lesions. Taken together, our proposed investigations will unravel the overall architecture of lymphostatin and the interplay between its functional modules. We expect that lymphostatin is modulated by simple structural changes and auto-proteolysis to activate or deactivate certain functions making it a multi-purpose tool. A detailed structural knowledge on these mechanisms will be essential for interfering with the action of lymphostatin and thus reducing the devastating effects of EHEC and EPEC infections in future.