# Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure
## 4 Filament
### Filament Cap  
Originally designated as a HAP2, FliD is localized at the tip of the filament where several copies of it assemble into a capping structure. The presence of the cap prevents the leakage of flagellin that is continuously exported through the flagellar type III secretion system into the filament channel and facilitates its insertion at the growing end of the filament. Early cryo-EM studies of the _Salmonella_ cap showed that it is a stool-like pentameric structure with the flat star-shaped head region and five legs positioned almost at the right angle. The head region is 145 A in diameter and the height of the cap is 125 A. While they vary in size, FliD s from different bacteria have conserved N- and C-terminal regions predicted to be coiled-coils and are largely unstructured in the monomeric state [93]. The first high-resolution structure of the _P. aeruginosa_ FliD showed that this 50 kDa protein has three distinct domains: a highly flexible D1 domain that corresponds to the leg domains seen in the cap structure, and compact D2 and D3 domains that comprise the head [94]. Much as in flagellin, a continuous sequence in the middle of the polypeptide folds into D3 while domains D1 and D2 consist of N- and C-terminal portions of the chain relative to D3. Domains D2 and D3 are each made of two antiparallel \(\beta\)-sheets, while D1 could not be resolved with confidence apart from one helix. The structure of the _E. coli_ FliD offered more insight into D1, showing that it is a helical bundle of at least four helices with a \(\beta\)-hairpin similar to the flagellin D1 domain, although approximately 50 terminal residues from both sides were not present in the construct (Figure 3a,c) [95]. These structures, together with the ones from _Salmonella_ and _Serratia marcescens_[95, 96], indicated that the structural organization of the capping proteins among different bacteria is preserved. However, not all FliD s are of the same size. For example, the FliD of _H. pylori_ is 74 kDa and the crystal structure revealed that apart from domains D1-3 present in the previously studied proteins, it has two additional domains, D4 and D5, both of which reside in the head region with similar overall folds to D2 and D3 (Figure 3b) [97]. These domains may be related to the specific role FliD plays as a virulence factor during _H. pylori_ infection; stronger antigenicity of D4 and D5 further supports this [97]. Once the hook and the junction are formed, FliD forms the filament cap, promoting the polymerization of FliC. The oligomeric state of the filament cap is still an open question. Initial characterization of the recombinant FliD of _Salmonella_ showed the existence of decamers in solution, but the combination of cross-linking experiments, analytical ultracentrifugation and electron microscopy revealed that a pentamer is the functional unit, decamers being the product of a non-physiological cap dimerization. This was generally also accepted as the case for other bacteria, and the recent cryo-EM structure of the pentameric C. _jejuni_ cap supports it [98]. However, both _Pseudomonas_ and _E. coli_ FliD s crystallized as hexamers and _Serratia_ FliD as a tetramer (Figure 3d) [94, 95, 96]. Moreover, tetramers and pentamers of _Pseudomonas_ FliD and tetramers of _Salmonella_ FliD were observed in vitro [94, 98]. While these stoichiometries could arise due to non-physiological conditions, in the case of _P. aeruginosa_ hexamers were also detected in vivo [94]. The mechanism of cap-filament interaction is yet to be explained. During filament elongation, new flagellin molecules arrive at the tip continuously. This would require high mobility of the cap and continuous conformational changes of the D1 domains, most probably independently from one another, in order to accommodate arriving FliC subunits, all while staying firmly attached to the filament.