**6.2 T3SS needle and pilus proteins**

The major extracellular T3SS component is the needle with a length of 60 nm and an external diameter of 7 nm for animal pathogens; a much longer structure (up to 2μm) named the Hrp pilus is the needle counterpart in phytopathogenic bacteria (Barrett et al., 2008; Cordes et al., 2003; He and Jin, 2003; Alfano & Collmer, 1997; Roine, 1997). The needle

structure for this motif (Trost et al., 2009). When the Protein Data Bank was searched for GxxxG repeats similar in length to those found in FliH, no helices containing more than three contiguous glycine repeat segments were found implying that long GxxxG repeats

Fig. 3. 3D-structures of T3SS proteins with significant coiled-coil content and their locations. The structure of the intrinsically disordered HrpO is based on SAXS data (Gazi et al., 2008).

The major extracellular T3SS component is the needle with a length of 60 nm and an external diameter of 7 nm for animal pathogens; a much longer structure (up to 2μm) named the Hrp pilus is the needle counterpart in phytopathogenic bacteria (Barrett et al., 2008; Cordes et al., 2003; He and Jin, 2003; Alfano & Collmer, 1997; Roine, 1997). The needle

are presumably quite rare in nature.

**6.2 T3SS needle and pilus proteins** 

appears to play a major role in host sensing and signal transmission from the distal to the basal end of T3SS (Deane et al*.*, 2006). The needle structures are formed through the helical assembly of multiple copies of a small α-helical protein. Along the needle axis runs a narrow (2.5 nm) conduit which is used for the passage of needle components, tip proteins, translocators and effectors, whereby a partially unfolded form of the substrate is required. Structures are available (Wang et al., 2007; Deane et al., 2006; Zhang et al., 2006) for three needle components from animal pathogens: MxiH (*S. flexneri*), BsaL (*B. pseudomallei*) and PrgI (*S. typhimurium*). These structures are highly α-helical, with a central coiled-coil which is essential for needle assembly. Outside this coiled coil, all three proteins have highly mobile N-termini and C-termini, although these regions may retain some degree of helical structure in solution (Blocker et al., 2008). The sequences of the coiled-coil parts of the needle proteins show strong similarities, suggesting that they all share a common fold and pattern of interactions (Wang et al., 2007; Zhang et al., 2006). Analysis of structures and sequences of needle components from various pathogens suggests that the majority has a propensity for structural disorder (Table 2). An analysis of needle/pilus components predicts a mean overall disorder of approx. 30%. The predicted disorder has been confirmed by CD and thermal unfolding studies of MxiH, BsaL and PrgI which reveal that under conditions resembling the physiological ones, all three C-terminally truncated proteins adopt a molten globule-like state; at temperatures above 37o C their tertiary structure collapses while the secondary structure is largely retained (Barrett et al., 2008); this behaviour is strongly reminescent to the one observed for the cytoplasmic HrpOSctO protein (Gazi et al., 2008). A partially unfolded state of the needle components could be functionally important e.g. for transversing the needle channel and for the extracellular assembly. Signal transmission for host cell sensing is suspected to utilize the flexibility of needle subunits (Deane et al., 2006).

The major subunits of the Hrp pilus (HrpA) are generally predicted to be almost entirely αhelical, with the exception of the *Pseudomonas syringae* species, for which the 50 N-terminal amino acids are predicted to contain β-strands (He and Jin, 2003; Koebnik, 2001). The major subunits of the Hrp-dependent pili, like the needle structural proteins, are all small proteins (of 6 to 11 kDa), but their sequences are surprisingly hypervariable, even within *P. syringae* pathovars. This hypervariability may reflect the evolutionary adaptations to evade plant defense systems. The predicted secondary structures of the major pilus subunits however, are remarkably similar, almost entirely α-helical. Insights into the structure of Hrp pilus components have been obtained from the recent investigation of the HrpA protein from the *P. syringae* pv. phaseolicola T3SS pilus (Kotzabasaki & Kokkinidis, unpublished). The Cterminal part of the 11 kDa protein is responsible for the assembly of multiple HrpA copies in the pilus (Roine, 1997). No chaperons for HrpA have been identified. The secondary structure of HrpA is predicted to be highly α-helical, with a propensity for coiled-coil formation in its functionally important C-terminal region. Surprisingly, experimental characterization of the HrpA protein using CD, Raman, FTIR and SAXS provides strong evidence that HrpA does not adopt a helical structure, but rather a highly disordered state with β-strand features. High resolution transmission electron microscopy (TEM) of purified HrpA samples reveals a pronounced propensity for polymerization and formation of two types of fibrils with nano-to micro scale features, one of which has comparable geometrical parameters with the Hrp pilus.


Fig. 4. Predicted coiled-coil regions and structural disorder for the HrpO/FliJ/YscO family. Disordered segments (predicted by FoldIndex) are colored red, coiled-coil domains cyan. All sequences were predicted to be almost entirely α-helical, which was confirmed for HrpO (Gazi et al., 2008), FliJ (Ibuki et al., 2011) and CT670 (Lorenzini et al., 2010). Sequences are drawn to scale, and vary from 166 (HrpD) to 125 residues (SsaO).

Fig. 5. T3SS proteins involved in coiled-coil interactions: (A) FliJ (Ibuki et al., 2011) from *S. typhimurium*, (B) CT670 SctO (Lorenzini et al., 2010) from *Ch. trachomatis* colored from N- (blue) to C-terminus (red). The dimeric FliT of *S. enterica* sv. typhimurium (C) and *Bordetella bronchiseptica* (D), each monomer differently coloured. In (C) the C-terminal helix adopts a different conformation in each monomer (Imada et al., 2010). (E) The *Y. enterocolitica* SycD (Buttner et al., 2008) which is recognized by YscO SctO (Evans & Hughes, 2009).

#### **6.3 T3SS effector flexibility and coiled-coil propensity**

Approximately a third of the T3SS effector structures known forms regular coiled-coils with knob-into-holes packing, while several others exhibit short heptad repeat patterns in their sequences which give rise to coiled-coil interactions and short, distorted α-helical

Fig. 4. Predicted coiled-coil regions and structural disorder for the HrpO/FliJ/YscO family. Disordered segments (predicted by FoldIndex) are colored red, coiled-coil domains cyan. All sequences were predicted to be almost entirely α-helical, which was confirmed for HrpO (Gazi et al., 2008), FliJ (Ibuki et al., 2011) and CT670 (Lorenzini et al., 2010). Sequences are

Fig. 5. T3SS proteins involved in coiled-coil interactions: (A) FliJ (Ibuki et al., 2011) from *S. typhimurium*, (B) CT670 SctO (Lorenzini et al., 2010) from *Ch. trachomatis* colored from N- (blue) to C-terminus (red). The dimeric FliT of *S. enterica* sv. typhimurium (C) and *Bordetella bronchiseptica* (D), each monomer differently coloured. In (C) the C-terminal helix adopts a different conformation in each monomer (Imada et al., 2010). (E) The *Y. enterocolitica* SycD

Approximately a third of the T3SS effector structures known forms regular coiled-coils with knob-into-holes packing, while several others exhibit short heptad repeat patterns in their sequences which give rise to coiled-coil interactions and short, distorted α-helical

(Buttner et al., 2008) which is recognized by YscO SctO (Evans & Hughes, 2009).

**6.3 T3SS effector flexibility and coiled-coil propensity** 

drawn to scale, and vary from 166 (HrpD) to 125 residues (SsaO).

bundles: A YpkA subdomain folds in two 3-helical bundles (Prehna et al*.*, 2006). Coiledcoils are also observed in the N-terminal domains of YopH and SptP (Khandelwal et al., 2002; Stebbins and Galan, 2000) and in MxiC and YopN-TyeA (Deane et al., 2008; Schubot et al., 2005). The *S. flexneri* effector IpaH is a E3 ubiquitin ligase with a C-terminal which consists entirely of α-helical bundles and carries the catalytic activity for ubiquitin transfer (Singer et al., 2008). Helical bundle structures are also adopted by AvrPto (Wulf et al., 2004) and AvrPtoB (Dong et al., 2009), although both interact with their target protein kinase Pto via β-strand addition (Xing et al., 2007). AvrPto (PDB ids: 1R5E, 2QKW) displays considerable structural plasticity which is consistent with its predicted increased flexibility (Table 1). In a recent analysis, 49% of the *S. typhimurium* effectors have been predicted to posses at least one coiled-coil domain which enhances membrane association in mammalian cells (Knodler et al., 2011).

Apart from the frequent occurrence of coiled-coils, a further common feature among T3SS effectors are disorder effects established both as localized disorder of their extreme Nterminal peptide where the secretion signal resides, or frequently as an overall structural disorder. Usually, the 15-20 N-terminal residues of effectors are highly disordered. Truncation of N- and C-terminal residues was necessary for the NMR study of the AvrPto effector (Wulf et al*.*, 2004), while in the crystal structure of the AvrPto-Pto kinase complex 28 N-terminal residues are missing (Xing et al*.*, 2007). In the case of ExsE the N- terminal fifteen residues had to be omitted for crystallization and structure determination (Vogelaar et al., 2010). Although, the full length AvrB and AvrPphF ORF2 were crystallized, electron density was not observed for the 27 N-terminal residues due to disorder (Lee et al., 2004; Singer et al., 2004). The N-terminal region of the SipA effector in the complex with the chaperone InvB is highly disordered (Lilic et al., 2006). In the CBD of effectors bound to class IA chaperones, there is a prevalent localized disorder for the part of the effector that crosses the interface of the dimeric chaperone; thus, this part of the effector cannot be modelled, as can be seen not only for SipA but also for ExsE and YopN (Vogelaar et al., 2010). Moreover, a large majority (~75%) of *P.syringae* pv. tomato effectors show a significant propensity for structural disorder in the region of their 50 N-terminal residues based on the ratio of ordervs. disorder- promoting residues (selected effectors are shown in Table 1) which has an average value of 0.45 (M. Kokkinidis, unpublished). Other secreted proteins have an average value of 0.50. The ratio for cytoplasmic T3SS proteins is 0.55; the average ratio in proteomes is 0.58 based on the amino acid frequencies of order- and disorder-promoting residues (Brooks et al., 2002); significantly lower values, as in the case of the N-termini of T3SS effectors, indicate a propensity for disorder. The only structured N-terminal region of a T3SS effector is that of YopH. The 129 residue N-terminal domain has two functions: the first 70 residues contain the CBD domain for chaperone SycH, while the full 129 N-terminal domain binds to phosphotyrosine-containing proteins and adopts an overall globular fold (Khandelwal et al., 2002). The N-terminal region of VirA is partially disordered (Davis et al., 2008). Apart from the N-terminal disorder, a propensity for overall disorder is predicted for T3SS effectors, which may reflect an increased structural flexibility. An average value of 35% of disordered residues is predicted by FOLDINDEX for the T3SS effectors of *P. syringae* pv. tomato DC3000, which is to be contrasted with an average value of 28% for cytoplasmic T3SS proteins, if the extensively disordered members of the HrpO/FliJ/YscO family (some of which could be classified as IDPs) are excluded. For other secreted/putatively secreted T3SS proteins the values are 30-37%.
