Mirrashidi et al. (2015), demonstrated an in vitro interaction between IncE and the SNX5 and SNX6 PX domains. To confirm their direct association we assessed the binding affinities using isothermal titration calorimetry (ITC) (Figure 2A; Table 1). Initial experiments with the human SNX5 and SNX6 PX domains showed robust interactions with the IncE C-terminal domain (residues 107–132). The affinities (Kd) for SNX5 and SNX6 were essentially indistinguishable (0.9 and 1.1 µM respectively), but we detected no interaction with the PX domain of SNX1 confirming the binding specificity. The PX domains of SNX5 and SNX6 possess a helix-turn-helix structural insert (Koharudin et al., 2009), which is not found in any other SNX family members except for SNX32 (Figure 2B), a homologue expressed primarily in neurons (BioGPS (Wu et al., 2009)). Confirming a common recruitment motif in the SNX5-related proteins, ITC showed a strong interaction between IncE and the SNX32 PX domain similar to SNX5 and SNX6 (Kd = 1.0 µM) (Figure 2A; Table 1), and SNX32 was robustly recruited to inclusion membranes in infected cells (Figure 1B; Figure 1—figure supplement 1A). Overall, our data indicates that a common structure within the SNX5, SNX6 and SNX32 PX domains is required for IncE interaction.10.7554/eLife.22311.007Figure 2.IncE from C. trachomatis binds the PX domains of SNX5, SNX6 and SNX32.(A) Binding affinity between IncE peptide (residues 107–132) and SNX PX domains by ITC. Top panels show raw data and lower panels show normalised integrated data. See Table 1 for the calculated binding parameters. Truncation analyses of the IncE peptide by ITC are shown in Figure 3, Table 2. (B) Sequence alignment of human SNX1, SNX5, SNX6 and SNX32 PX domains. Conserved residues are indicated in red. Side-chains that directly contact IncE in the crystal structure are indicated with black circles. Mutations that block IncE binding are highlighted with red triangles, and mutations that do not affect binding indicated with green circles. Secondary structure elements derived from the SNX5 crystal structure are indicated above. (C) Sequence alignment of IncE from C. trachomatis and putative homologues from C. muridarum and C. suis. IncE side-chains that directly contact SNX5 in the crystal structure are indicated with black circles. Mutations that block SNX5 binding are highlighted with red triangles, and mutations that do not affect binding indicated with green circles. Predicted transmembrane regions are indicated and C-terminal IncE sequences that form β-strands in complex with SNX5 are shown.DOI: http://dx.doi.org/10.7554/eLife.22311.00710.7554/eLife.22311.008Table 1.Thermodynamic parameters of IncE binding to SNX PX domains*.DOI: http://dx.doi.org/10.7554/eLife.22311.008Sample cellTitrantKd (µM)△H (kcal/mol)T△S (kcal/mol)△G (kcal/mol)NSNX5 PXIncE peptide†0.95 ± 0.07−6.9 ± 0.3−1.9 ± 0.05−8.2 ± 0.011.01 ± 0.01SNX6 PXIncE peptide1.13 ± 0.08−5.0 ± 0.9−3.0 ± 1−8.0 ± 0.071.01 ± 0.08SNX32 PXIncE peptide1.15 ± 0.07−6.9 ± 0.4−1.3 ± 0.8−8.2 ± 0.41.06 ± 0.005SNX1 PXIncE peptideNo binding*Values are the mean from three experiments ±SEM.b.†IncE synthetic peptide sequence PANGPAVQFFKGKNGSADQVILVTQ.