Structural and functional investigation of the Paraquat inducible pathway

Cooper, Benjamin Francis ORCID: 0000-0002-4023-9011 (2023). Structural and functional investigation of the Paraquat inducible pathway. University of Birmingham. Ph.D.

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Abstract

Originally proposed to facilitate cell invasion, Mammalian Cell Entry (MCE) domains have subsequently been implicated in the transport of hydrophobic substances. Indeed, multiple phylogenetic and biochemical studies have proposed members of the MCE protein superfamily to traffic lipid species. The E. coli Mla pathway, itself containing the MCE protein MlaD, presents the first proteinaceous system facilitating intermembrane GPL trafficking. E. coli contains two further MCE proteins, YebT and PqiB, each residing in unique operons which have become of principal interest as putative Gram-negative GPL transporters.

Here we focus upon the Pqi pathway, a tripartite macromolecular complex, encoded by a single PqiA-C operon, anticipated to span the periplasm allowing the transport of lipid species between diderm membranes. We present X-ray structures of the native PqiC lipoprotein and that of a soluble PqiC\(^1\)\(^7\)\(^-\)\(^1\)\(^8\)\(^7\) construct revealing a potentially biologically relevant octameric ring assembly and high-resolution side chain information respectively. We also provide the first structure of native PqiB, via cryo-EM, allowing comparison to the existing soluble PqiB\(^3\)\(^9\)\(^-\)\(^5\)\(^4\)\(^6\) structure and demonstrating flexibility within the pore lining loop regions. Finally, we utilise phenotypic complementation assays to identify residues within PqiA and PqiB essential for Pqi function and provide evidence for a protonation dependent PqiA mechanism.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):