Structural Studies of Lymphostatin

• Applicant: Professorin Dr. Bettina Böttcher
• Subject Area: Structural Biology
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 428774170

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.

• DFG Programme: Research Grants