# Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure
## 5 Protein Synthesis, Export and Filament Elongation
### Chaperones
#### 5.2.1 FliS--Flagellin Chaperone  
FliS acts as a flagellin-specific T3 SS chaperone of FliC, preventing premature folding and inappropriate interaction of newly synthesized flagellin subunits in the cytosol, thus facilitating its export and polymerization upon completion of the HBB assembly. Yeast two-hybrid assays indicated that the C-terminal disordered region of flagellin is essential for FliS binding and, accordingly, spontaneous mutations causing flagellin accumulation in the cytoplasm map to the C-terminal region of FliC [132]. Thermodynamic experiments indicated that FliS does not function as an anti-folding factor keeping flagellin in a secretion-competent conformation. Instead, FliS binding stabilizes the flagellin conformation through formation of the \(\alpha\)-helical secondary structure in the last 40 C-terminal residues of FliC (residues 454-494) [105; 133; 134]. The first crystal structure of a flagellar chaperone, _Aquifex aeolicus_ FliS (AaFliS), revealed a novel, mainly \(\alpha\)-helical fold, different from those of the T3 SS chaperones [134]. The structure of _AaFliS chaperone_ in complex with a C-terminal fragment of its cognate flagellin (PDB: 1 ORY) shows a monomer-to-monomer interaction. However, it was also reported that FliS binds in a 2:1 stoichiometry to the C-terminal region of flagellin to completely prevent the premature polymerization of newly synthesized flagellin molecules, as a 1:1 FliS to FliC molar ratio was able to prevent FliC polymerization only partially. Intriguingly, no stable FliS-FliC complex could be detected in _S. typhimurium_ cell extracts subjected to gel-filtration chromatography, suggesting that this interaction may be transient in vivo. Such rapid chaperone dissociation would favor the subsequent export of FliC [135]. The interaction between FliS chaperone and FliA is a key step preceding the efficient transfer of FliC to the platform of the flagellar type III export apparatus for a rapid export during flagellar filament assembly [136; 137; 138]. Filament proteins in complex with their cognate chaperones bind to a highly conserved hydrophobic pocket of FlhAC to promote unfolding and protein translocation by the protein export apparatus. It was predicted that different binding affinities of FlhAC for the chaperone substrate complexes may be the key to defining the correct order of protein export among the filament-type proteins [139]. Recently, a structure of the ternary complex formed by FliC, FliS and the export gate protein FlhA revealed that FliC does not interact directly with FlhA (Figure 5). Instead, the presence of FliC induces a binding-competent conformation of FliS that exposes the motif which is specifically recognized by FlhA [140]. Moreover, SAXS and HDX-MS experiments showed the formation of a heterotrimic FliC-FliS-FliW complex that interacts with FlhA suggesting that FliS and FliW are released during flagellin export. FliW and FliS bind to opposing interfaces located at the N- and C-termini of flagellin, respectively, and these proteins seems to synchronize the production of flagellin with the capacity of the T3 SS to secrete flagellin [141]. FliS plays an additional role in suppressing the secretion of FlgM in _Salmonella_. The loss of FliS results in a short filament phenotype despite high expression levels of FliC, which is explained by the increase in the secretion level of FlgM [136]. Bypass mutants have been isolated from a _Salmonella_\(\Delta\)_fliS_ mutant, and all those mutations were identified in FliC [142, 143]. [PERSON] et al. and [PERSON] et al. investigated the direct interaction between FliS and FlgM from _S. typhimurium_ and _Yersinia pseudotuberculosis_, respectively [135, 144]. Using a number of different approaches, they showed that these proteins specifically interact to form a 1:1 complex, and that this interaction protects FlgM from proteolysis. FliS acts as a negative  
Figure 5: Bacterial flagellar filament proteins and their cognate specific chaperone form complexes with FlhA for filament exportation/synthesis.