**6.1 Cytoplasmic proteins**

Several cytoplasmic T3SS proteins exhibit a significant coiled coil propensity and intrinsic disorder (Table 1, Fig. 3, 4). Evidence from some cytoplasmic proteins (see section 6.1.1) suggests that these properties might be essential elements in the establishment of key protein-protein interaction networks required for T3SS function (Gazi et al., 2008).

### **6.1.1 The SctO family (HrpO/FliJ/YscO homologs)**

The most extensive heptad repeat pattern occurs in the HrpO/FliJ/YscO family of T3SS proteins (Gazi et al., 2008). Despite the absence of significant homologies, the family members share specific characteristics, e.g. increased propensity for coiled coil formation and intrinsic disorder (Gazi et al., 2008). The extreme flexible nature of HrpOSctO from *Pseudomonas syringae* pv. phaseolicola (Gazi et al., 2008), a property shared with FliJ from *S. typhimurium,* has prevented its crystallization and determination of its 3D-structure by X-ray crystallography. A variant form of the FliJ protein from *S. enterica* sv. typhimurium was crystallized however (Ibuki et al., 2009), and its structure was found to be remarkably similar (Fig. 5) to that of the two-stranded α-helical coiled-coil part of the γ subunit of F0F1- ATP synthase (Ibuki et al., 2011). A similar coiled coil structure (Fig. 5) consisting of two long α-helices was also reported for the crystal structure of the CT670SctO protein (a YscO homolog) from *Chlamydia trachomatis* (Lorenzini et al., 2010).


Table 2. Disorder analysis for T3SS proteins of known 3D-structures with coiled-coil content exceeding 30%. The overall protein disorder was calculated from sequence data using the FoldIndex program with a window of 21 residues.

Small angle X-ray scattering (SAXS) and circular dichroism (CD) characterization of HrpOSctO from *P. syringae* pv. phaseolicola revealed a high α-helical content with coiled-coil characteristics and molten globule-like properties (Gazi et al., 2008). HrpOSctO like its flagellar counterpart FliJ is essential for export, but its function remains obscure. HrpOSctO interacts, probably via intermolecular coiled-coil formation, with HrpE, a highly α-helical T3SS protein which belongs to the HrpE/FliH/YscL family. FliH, the flagellar counterpart of HrpE is a regulator of the FliI ATPase (Lane et al., 2006). Evidence from HrpOSctO and its analogs in various flagellar or non-flagellar T3S systems suggests that the extreme flexibility (Fig. 4) and propensity for coiled-coil interactions observed in members of the HrpO/FliJ/YscO family might be important factors for increased interactivity and the establishment of functional protein-protein interaction networks in T3SS. This is consistent with the observation that several members of the HrpO/FliJ/YscO family were found to interact with other cytosolic T3SS components or self-associate via coiled-coil interactions: The flagellar FliJ protein, a key player in a chaperone escort mechanism that recruits unloaded chaperones for the minor filament-class subunits of the filament cap and hookfilament junction substructures (Evans et al., 2006) binds to the same chaperone site as the cognate export substrate of the chaperone, albeit with a much lower affinity. Similarly, YscOSctO from *Yersinia enterocolitica* and InvISctO from *Salmonella typhimurium* do not bind to export substrates but recognize a subset of export chaperones that are specialized to deliver the T3SS translocators to the export apparatus (Evans & Hughes, 2009). In all of these cases the interaction partners of the HrpO/FliJ/YscO family members exhibit a very high αhelical/coiled-coil content (Fig. 5). FliJ was also found to interact with structural cytoplasmic components of the T3SS like the FliMSctQ protein, even in the absence of FliH, suggesting a docking mechanism for export substrates, chaperones and the ATPase to the T3SS machinery (Gonzalez-Pedrajo et al., 2006). The CT670SctO protein exists in monomeric and

*Pseudomonas syringae* pv. phaseolicola (Gazi et al., 2008), a property shared with FliJ from *S. typhimurium,* has prevented its crystallization and determination of its 3D-structure by X-ray crystallography. A variant form of the FliJ protein from *S. enterica* sv. typhimurium was crystallized however (Ibuki et al., 2009), and its structure was found to be remarkably similar (Fig. 5) to that of the two-stranded α-helical coiled-coil part of the γ subunit of F0F1- ATP synthase (Ibuki et al., 2011). A similar coiled coil structure (Fig. 5) consisting of two long α-helices was also reported for the crystal structure of the CT670SctO protein (a YscO

**NEEDLE CHAPERONES TIP**  MxiH 52 SycD 12 LcrV 45 PrgI 21 PscE 0 IpaD 41 BsaL 30 PscG 7 BipD 26 YscE 27 EspA 6

Table 2. Disorder analysis for T3SS proteins of known 3D-structures with coiled-coil content exceeding 30%. The overall protein disorder was calculated from sequence data using the

Small angle X-ray scattering (SAXS) and circular dichroism (CD) characterization of HrpOSctO from *P. syringae* pv. phaseolicola revealed a high α-helical content with coiled-coil characteristics and molten globule-like properties (Gazi et al., 2008). HrpOSctO like its flagellar counterpart FliJ is essential for export, but its function remains obscure. HrpOSctO interacts, probably via intermolecular coiled-coil formation, with HrpE, a highly α-helical T3SS protein which belongs to the HrpE/FliH/YscL family. FliH, the flagellar counterpart of HrpE is a regulator of the FliI ATPase (Lane et al., 2006). Evidence from HrpOSctO and its analogs in various flagellar or non-flagellar T3S systems suggests that the extreme flexibility (Fig. 4) and propensity for coiled-coil interactions observed in members of the HrpO/FliJ/YscO family might be important factors for increased interactivity and the establishment of functional protein-protein interaction networks in T3SS. This is consistent with the observation that several members of the HrpO/FliJ/YscO family were found to interact with other cytosolic T3SS components or self-associate via coiled-coil interactions: The flagellar FliJ protein, a key player in a chaperone escort mechanism that recruits unloaded chaperones for the minor filament-class subunits of the filament cap and hookfilament junction substructures (Evans et al., 2006) binds to the same chaperone site as the cognate export substrate of the chaperone, albeit with a much lower affinity. Similarly, YscOSctO from *Yersinia enterocolitica* and InvISctO from *Salmonella typhimurium* do not bind to export substrates but recognize a subset of export chaperones that are specialized to deliver the T3SS translocators to the export apparatus (Evans & Hughes, 2009). In all of these cases the interaction partners of the HrpO/FliJ/YscO family members exhibit a very high αhelical/coiled-coil content (Fig. 5). FliJ was also found to interact with structural cytoplasmic components of the T3SS like the FliMSctQ protein, even in the absence of FliH, suggesting a docking mechanism for export substrates, chaperones and the ATPase to the T3SS machinery (Gonzalez-Pedrajo et al., 2006). The CT670SctO protein exists in monomeric and

**disorder Protein % total** 

**disorder** 

homolog) from *Chlamydia trachomatis* (Lorenzini et al., 2010).

 YscG 39 CesA 66

FoldIndex program with a window of 21 residues.

**disorder Protein % total** 

**Protein % total** 

dimeric forms, with the monomeric form dominating at low protein concentrations. For selfassociation and dimer formation the involvement of coiled-coil interactions is predicted (Lorenzini et al., 2010). CT670SctO interacts with CT671SctP, a T3SS protein, with a predicted coiled-coil domain in its C-terminal region. CT671SctP is a homolog of the YscP protein which has been characterized as a molecular ruler and as a switch for T3SS substrate specificity in *Yersinia* species (Agrain et al., 2005). The two coiled-coil containing proteins CT670SctO and CT671SctP have been suggested to form a chaperone-effector-like pair with CT670SctO acting as chaperone (Lorenzini et al., 2010).

The HrpO/FliJ/YscO family members are encoded by genes located always downstream of the gene coding for T3SS ATPases (the SctN family of T3SS proteins which includes HrcN/FliI/YscN homologs); this implies a close connection between these proteins and the ATPase. In flagellar T3SS the FliI protein is an ATPase that has extensive structural similarity to the α- and β- subunits of the FoF1-ATP synthase (Imada et al., 2007), while also the structure of FliJ from *S. enterica* sv. typhimurium (Fig. 4, 5) is remarkably similar to that of the two-stranded α-helical coiled-coil part of the γ-subunit of FoF1-ATP synthase (Ibuki et al., 2001). FliJ promotes the formation of FliI hexamer rings by binding to the center of the ring in a similar way to the γ-subunit penetrating into the central channel of the α3β3 ring in FoF1-ATPase. Moreover, the HrpE/FliH/YscL family of proteins (interaction partners of the HrpO/FliJ/YscO family) are distant homologs to both β- and δ- subunits of the FoF1-ATP synthase (Pallen et al., 2006). In flagellar systems the docking of the ATPase to the T3S machinery is mediated by the FliJ/FliH pair (Minamino et al., 2009). These results strongly suggest that T3SS and F- and V-type ATPases share a similar mechanism and an evolutionary relationship. It is thereby striking that extensive coiled-coil domains (e.g. FliJ, FliH) have been conserved between the two systems.

Overall, the above remarkable findings support our earlier suggestions (Gazi et al., 2008, 2009) that T3SS proteins, and in particular members of the SctO family, with long disordered/flexible coiled coil structures occupy node positions in the T3SS interactome, being capable of interacting with different partners and possess various roles in the secretion mechanism. These roles are to a large extent poorly understood and remain to be elucidated experimentally.
