**9. Acknowledgment**

24 Biochemistry

The combination of coiled-coiled interactions and structural plasticity are frequently essential prerequisites for the establishment of interaction networks within T3SS, as exemplified by the interactions of proteins of the HrpO/FliJ/YscO family with members of the HrpE/FliH/YscL family (Gazi et al., 2008), the SipD/PrgI (Lunelli et al., 2011; Rathinavelan et al., 2011) or the CT670/CT671 interaction (Lorenzini et al., 2010). In addition, the assembly of the T3SS supramolecular structures frequently requires a combination of coiled-coils and conformational flexibility: T3SS needle assembly occurs through the stepwise polymerization of a major subunit (e.g. MxiH, BsaL and PrgI) via a flexible or partially disordered C-terminal helix which exhibits a propensity for coiled-coil interactions. For the IDP HrpA polymerization into pilus-like fibrils has been observed, although no experimental evidence for the involvement of coiled-coil interactions could be

The propensity for disorder is frequently reflected the amino acid composition of T3SS protein sequences. The vast majority N-terminal sequences of T3SS effectors and other secreted proteins exhibits specific biases (Table 1) in their composition with respect to order- and disorder-promoting residues (Dunker et al., 2002; Uversky, 2010), from which a disorder propensity can be predicted, usually in agreement with experimental observations. Interestingly, these disorder-associated biases (as reflected in the ratio of order- vs. disorder- promoting residues), result in sequence preferences for the N-termini which are similar to those determined for T3SS effectors from various bacterial species (Greenberg and Vinatzer, 2003). The structural disorder of the N-termini may thus play a role as a secretion signal, a suggestion made earlier by Akeda & Galan (2005) and confirmed by subsequent analyses (Gazi et al., 2009). However, as N-terminal structural disorder does not ensure specificity of substrate recognition (e.g. the cytoplasmic HrcQB protein is predicted to possesses a highly flexible N-terminus), it may be assumed that Nterminal flexibility could be one of multiple secretion signals (Marlovits et al*.*, 2006), with other signals, e.g. chaperones, ensuring specificity. Analysis of effectors and other secreted/non-secreted T3SS components strongly suggests that the overall disorder of T3SS proteins is a further parameter strongly correlated with secretion (Table 1, 2). Flexible or disordered T3SS domains could facilitate rapid unfolding which is necessary for secretion. Both N-terminal and overall flexibility might be thus considered in prediction algorithms for the identification of universal T3SS effectors signatures; this would complement recent efforts based on machine learning approaches (Arnold et al., 2009; Samudrala et al., 2009). Interestingly, sequence stretches with coiled-coils propensities are suitable tertiary motifs to provide the necessary flexibility which is proposed to be associated with secretion. In fact, coiled-coil proteins are frequently viewed as a specific set of intrinsically disordered proteins (Gaspari & Nyitray, 2011) and occasionally they have been observed to display molten globule characteristics (Glykos & Kokkinidis, 2004). A further advantage of coiled-coils might be associated with specific features of their disordered state: As shown in the case of the HrpO protein (Gazi et al., 2008), proteins exhibiting coiled-coil propensity are capable of adopting highly nonglobular conformations, while maintaining a considerable α-helical content. The geometrical dimensions of such non-globular helical conformations permit passage through the narrow needle/pilus channel if the appropriate secretion signal is present. It is intuitive to assume that after passing this conduit, such preformed and foldingcompetent helices encompassing a few turns may form a nucleation site which promotes

obtained, despite the high α-helical content predicted by sequence analysis.

Work was partially funded by the GSRT, Joint Research and Technology Programmes for Greece-France (2010-11). Support for access and use of the SOLEIL synchrotron for SAXS studies is acknowledged. V.E.F is supported by a Marie Curie Reintegration grant. Figure 1 was produced with UCSF Chimera, University of California, San Francisco
