FliI has a non-conserved N-terminal region (amino acids 1-150) which may be important for mediating protein-protein interactions, a catalytic domain (amino acids 150-329), containing a conserved P loop, Walker A and B domains, and a C-terminal non-conserved domain (amino acids 329-434) of unknown function.

FliI has a 34 percent sequence similarity to CdsN, the C. pneumoniae T3S ATPase, and 36 percent similarity with FliI from Salmonella. The active domain of FliI has the most similarity to its paralogs and orthologs, while the N- and C-terminal regions have the lowest amount of similarity (Figure 1). Figure 1 Sequence conservation WH-4-023 order of FliI from C. pneumoniae with C. trachomatis and Salmonella. Sequence alignment (ClustalW) of the full length FliI protein from C. pneumoniae, C. trachomatis, and Salmonella. Asterisk refers to identical amino acids, a double dot refers to a conserved substitution and a single dot refers to a semi-conserved substitution. Outlined is the conserved P loop region in the Walker A domain. FlhA (Cpn0363) is 583 amino acids in length with a predicted molecular mass of 65.6 kDa and a pI of 5.60. The FlhA paralog in C. pneumoniae is the T3S protein CdsV. FlhA has seven predicted transmembrane regions in the N-terminal half of

the protein (FlhA1-308), while the C-terminal half of the protein is predicted to be cytoplasmic (TMpred). FlhA from C. pneumoniae has 21 percent sequence orthology with FlhA from Salmonella. FliF (Cpn0860) Autophagy Compound Library solubility dmso is 342 amino acids in length with a predicted molecular mass of 38.2 kDa and a pI of 9.5. The FliF paralog in C. pneumoniae is the T3S protein CdsJ. FliF has two predicted TM regions, one located near the N-terminus and one located near the C-terminus.

FliF from C. pneumoniae is only 15 percent similar to FliF from Salmonella. Expression and ATPase activity of FliI FliI has significant similarity with many characterized Meloxicam ATPases, and this led us to explore the ATPase activity of this protein. Purified GST tagged FliI was tested for its ability to hydrolyze ATP using the malachite green binding assay. Figure 2A shows that GST-FliI was essentially free of contaminating proteins by SDS-PAGE and anti-GST Western blot (left) or Coomassie blue stain (right). GST-FliI hydrolyzed ATP in a dose- and time-dependant manner at a rate of 0.15 ± .02 μmol min-1 mg-1 (Figure 2B i and ii, diamonds). This level of activity is comparable to other flagellar ATPases as well as the T3S paralog, CdsN [16, 41, 42]. ATPase activity of GST-FliI peaked at 37°C, and at a pH of 8.0 (Figure 2 iii and iv). Another GST-tagged protein, GST-CopN, purified in the same manner as GST-FliI had negligible ATPase activity (Figure 2B i and ii, squares). Figure 2 Expression, purification, and optimal conditions for the time- and dose-dependant ATPase FliI.