**6. The occurrence of coiled-coils and intrinsic disorder in T3SS proteins**

Analyses of T3SS protein sequences (Table 1) reveal an unusually frequent occurrence of predicted heptad repeats, which is indicative of a high propensity for coiled-coil formation (Delahay and Frankel, 2002; Pallen et al., 1997; Gazi et al., 2009; Knodler et al., 2011). Structural studies have confirmed the unsual prevalence of coiled-coils among T3SS proteins (Gazi et al., 2009; Ibuki et al., 2009; Lorenzini et al., 2010). In addition, coiled-coil interactions occur frequently in crystal structures of T3SS protein complexes, e.g. in the a macromolecular assembly TyeA-YopN that regulates type III secretion in *Yersinia pestis*  (Schubot et al., 2005) or in the complex of the filament protein EspA from the enteropathogenic *E. coli* T3SS with its chaperone CesA (Yip et al., 2005a). In a recent report, the interactions of the *Salmonella typhimurium* needle protein PrgI, an α-helical hairpin, with the tip protein SipD which comprises a long, central coiled coil (Rathinavelan et al., 2011) were studied using NMR paramagnetic relaxation enhancement. A specific region on the SipD coiled-coil was identified as the binding site for the α-helix of PrgI. Crystallographic studies of the PrgI-SipD complex have revealed coiled-coil interactions via the formation of an intermolecular 4-α-helical bundle structure (Lunelli et al., 2011). These studies also showed the importance of the structural flexibility of SipD (introduced by a π-bulge structure) in complex formation. Coiled-coil interactions of HrpO and FliJ with their cognate protein targets have been also reported (Gazi et al., 2008).

Disordered regions may be detected in protein structures determined by X-ray crystallography through missing electron density. Heteronuclear multidimensional NMR is a powerful tool for the characterization of protein disorder and provides direct measurement of the mobility of unstructured regions (Eliezer, 2007). Loss of secondary structure may be detected (among other methods) by far-UV CD (Kelly & Price, 1997) and Fourier transform infra-red spectroscopy (FTIR) (Uversky et al., 2000). Hydrodynamic parameters obtained from techniques such as gel filtration, SAXS (Gazi et al., 2008), dynamic and static light scattering provide information on whether a protein is unfolded since the unfolding results in an increase in protein hydrodynamic volume. The degree of globularity, which reflects the presence of a well-packed hydrophobic core may be estimated by a special analysis of small angle X-ray scattering (SAXS) data in form of a Kratky plot. Kratky plots are obtained by plotting *I(s)*x*s2* against *s* (scattering intensity: *I*; momentum transfer: *s*=4πsin(*θ*)/λ; 2*θ*: scattering angle; wavelength of X-rays: *λ*). They are used to judge the folding of the protein, as the shape of the curve is sensitive to the conformational state of the

Several algorithms have been developed to predict protein disorder on the basis of specific biochemical properties and biased amino acid compositions. These tools include PONDR (Romero et al., 2001; Peng et al., 2005), DisEMBL (Linding et al., 2003), IUPred (Dosztanyi et al., 2005), FoldUnfold (Galzitskaya et al., 2006) and PrDOS (Ishida & Kinoshita, 2007).

The main tool used in sections 6 and 7 for the *in silico* prediction of protein disorder from sequences is FoldIndex© (Prilusky et al., 2005). The propensity of N-termini of proteins for disorder was analyzed on the basis of their biased content of order-/disorder- promoting

Analyses of T3SS protein sequences (Table 1) reveal an unusually frequent occurrence of predicted heptad repeats, which is indicative of a high propensity for coiled-coil formation (Delahay and Frankel, 2002; Pallen et al., 1997; Gazi et al., 2009; Knodler et al., 2011). Structural studies have confirmed the unsual prevalence of coiled-coils among T3SS proteins (Gazi et al., 2009; Ibuki et al., 2009; Lorenzini et al., 2010). In addition, coiled-coil interactions occur frequently in crystal structures of T3SS protein complexes, e.g. in the a macromolecular assembly TyeA-YopN that regulates type III secretion in *Yersinia pestis*  (Schubot et al., 2005) or in the complex of the filament protein EspA from the enteropathogenic *E. coli* T3SS with its chaperone CesA (Yip et al., 2005a). In a recent report, the interactions of the *Salmonella typhimurium* needle protein PrgI, an α-helical hairpin, with the tip protein SipD which comprises a long, central coiled coil (Rathinavelan et al., 2011) were studied using NMR paramagnetic relaxation enhancement. A specific region on the SipD coiled-coil was identified as the binding site for the α-helix of PrgI. Crystallographic studies of the PrgI-SipD complex have revealed coiled-coil interactions via the formation of an intermolecular 4-α-helical bundle structure (Lunelli et al., 2011). These studies also showed the importance of the structural flexibility of SipD (introduced by a π-bulge structure) in complex formation. Coiled-coil interactions of HrpO and FliJ with their cognate

**6. The occurrence of coiled-coils and intrinsic disorder in T3SS proteins** 

protein targets have been also reported (Gazi et al., 2008).

**5.3 Experimental and** *in silico* **analysis of disordered domains** 

scattering molecules (Gazi et al., 2008).

residues (Dunker et al., 2002).

Predicted coiled-coil domains have been shown by mutagenesis to enhance membrane association of *Salmonella* T3SS effectors (Knodler et al., 2011). T3SS proteins and coiled-coil domains are frequently predicted to be structurally disordered (Table 1, 2). For many T3SS effectors disorder in their N-terminal region, as well as an increased overall flexibility have been also noted (Table 1, Gazi et al., 2009). In the following, these aspects of T3SS proteins will be elaborated with specific examples from various protein families. Structures of T3SS proteins with increased coiled-coil content are shown in Fig. 3


Table 1. Heptad repeats prediction and disorder analysis for selected proteins from the T3SS of *P. syringae* pv. tomato DC3000. Only proteins with coiled-coil content above 20% are given. For the AvrPto1 protein the crystalographically determined coiled-coil content is given in parentheses. The overall disorder was calculated using FOLDINDEX. N-terminal protein disorder calculations used Dunker's et al. (2002) definition of order-/ disorderpromoting residues. HrcQB does not include the disordered N-terminal domain.
