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Annotation, Biochemical and structural analysis of the Helicobacter pylori flagellum chaperone FlgN in complex with its binding partners: FlgK and FliI



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FlgN is a chaperone in motile bacteria that plays a crucial role in delivering the hook filament junction proteins FlgK and FlgL to the export apparatus during flagellum biogenesis. Deletion of FlgN results in a flagellum that terminates at the hook structure. The energy required for this process is thought to be provided by the flagellum-specific ATPase, FliI. Information available regarding FlgN interactions with substrates and export apparatus proteins FlhA and FliJ come from studies on S. typhi. Only indirect information is available on whether FlgN interacts with the FliI ATPase. Currently, no biochemical or structural information is available for FlgN from the proteobacteria, including C. jejuni or H. pylori. In this thesis, we will identify and study FlgN from H. pylori, a member of the proteobacteria and a human gastric pathogen that requires a functioning flagellum for infection. Here, I have used conservation of gene synteny, BLAST searches and multiple sequence alignments to demonstrate that the HP1120 gene encodes the FlgN chaperone. Using biochemical and biophysical techniques I have determined the stoichiometry of the interaction between H. pylori FlgN and FlgK to be 1:1 with very strong affinity. I have also shown that two of the three apo-FlgN structures available are domain swapped dimers with no substrate binding pocket. Using structural modelling, I have generated a working model for the unswapped H. pylori FlgN chaperone which clearly identifies the binding pocket for its substrates. I also show that FlgN and FliI interact strongly with 1:1 stoichiometry using biochemical and biophysical techniques. FliI is also known to interact with another flagellum chaperone FliT, and our work underscores important similarities between FlgN and FliT. Using mutagenesis, I have also provided evidence that the FlgN-FlgK interaction is not dominated by any one residue, but by a number of residues lining the binding pocket. Finally, I have solved the FlgK structure using crystallography which shows the presence of multiple Ca2+ binding sites which likely are integral for the structural stability of the protein. In conclusion, this work demonstrates a strong functional correlation between the FlgN and FliT flagellum chaperones.



Flagellum, T3SS, H. pylori, FlgN, chaperone



Doctor of Philosophy (Ph.D.)






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