Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.791735
Title: Structural studies of the type three secretion system export apparatus
Author: Kuhlen, Lucas
ISNI:       0000 0004 8503 4065
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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Abstract:
One of the most widespread bacterial secretion systems is the type three secretion system (T3SS), which is critical for the virulence of many pathogens and responsible for assembly of the bacterial flagellum. The virulence-associated T3SS is a molecular syringe that uses a highly conserved export apparatus (EA) to secrete effector proteins through a needle directly into the host cell cytoplasm. The flagellar T3SS secretes flagellar subunits through the EA which then self-assemble into a filament. Despite recent progress in the structural biology of T3SS, the highly conserved membrane domains of the five inner membrane proteins of the EA have evaded structural characterisation. Here, multiple cryo-EM structures of complexes of the three core EA proteins from both flagella (FliPQR) and injectisomes (SctRST) are presented, revealing a pseudohexameric, helical complex. Remarkably, the complex is made up of a series of long, highly kinked helical hairpins rather than the predicted canonical transmembrane helices. Many of the hydrophobic residues predicted to lie in transmembrane helices are instead buried at the interfaces between subunits, leaving only a small membrane domain. In the assembled T3SS this complex is encased in the basal body, above the plane of the inner membrane, and connects to the helical filament, whose helical parameters it matches. The membrane domain of the fourth export apparatus protein, FlhB/SctU, is shown to form a complex with FliPQR, and the complex structure suggests a mechanism of how interactions between FlhB and FliPQR could lead to opening of the complex. Finally, stable rings of full-length FlhA/SctV, the last EA protein, were prepared and cryo-EM confirmed the structure of the nonameric cytoplasmic domain. While little high-resolution information of the membrane domain was obtained, these studies provide a framework for future studies. Together these results reveal the structure of part of the secretion pathway and suggest that the core of the export apparatus templates the assembly of the helical filament.
Supervisor: Lea, Susan ; Robinson, Carol Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.791735  DOI: Not available
Keywords: Membrane Proteins ; Proteins--Structure
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