Finally we tested a series of IncE truncation mutants for their binding to the SNX5 PX domain (Figure 3A, B and C; Table 2). Synthetic peptides were used with single amino acids removed sequentially from the N and C-terminus to determine the minimal sequence required for binding. These experiments showed that the shortest region of IncE able to support full binding to SNX5 encompasses residues 110–131 (GPAVQFFKGKNGSADQVILVT), while a shorter fragment containing residues 113–130 (VQFFKGKNGSADQVILV) can bind to SNX5 with a slightly reduced affinity. While variations are observed across the different C. trachomatis serovars (Harris et al., 2012) the SNX5-binding sequence appears to be preserved in all detected variants (Figure 3D). A comparison with other chlamydial species suggests that IncE is not very widely conserved in this Genus, being clearly identifiable only in the closely related mouse pathogen C. muridarum and swine pathogen C. suis (Figure 2C). Residues required for binding to SNX5 are preserved in these IncE homologues, but whether SNX proteins are also recruited during infection by these other chlamydial species remains to be determined.10.7554/eLife.22311.009Figure 3.IncE residues 110–131 are sufficient for full recognition of the SNX5 PX domain.(A) Representative ITC experiments for truncated IncE peptides. These experiments were conducted using a single batch of SNX5 PX domain to minimize batch-to-batch protein variation. (B) Plots of the affinity constants for selected peptides to highlight the progressive loss of binding with N and C-terminal truncations. (C) Sequences of the truncated IncE peptides are given, with a qualitative indication of binding strength relative to the IncE_1 peptide containing residues 107–132. Full binding is indicated by ‘++’ reduced binding by ‘+’ and lack of binding by ‘−‘. All sequence information and their Kd values are given in Table 2. When compared to the reference ITC experiment the binding affinity of peptides was unaffected when the first three N-terminal residues were removed (IncE_2, IncE_3 and IncE_4) and gradually became weaker until IncE_7, after which binding was abolished. Results from IncE_6 are inconclusive due to the difficulty in successfully dissolving the peptides in buffer (n.d.). C-terminal truncations showed that IncE_14 and IncE_15 had similar high binding affinities to the reference, while the binding of IncE_16 and IncE_17 became progressively weaker and peptides shorter than IncE_17 showed no binding. This data indicates that the minimal IncE binding sequence retaining full SNX5 binding is GPAVQFFKGKNGSADQVILVT, and a shorter fragment VQFFKGKNGSADQVIL can bind to SNX5, albeit with a slightly reduced affinity. (D) Sequence alignment of IncE from different C. trachomatis serovars.DOI: http://dx.doi.org/10.7554/eLife.22311.00910.7554/eLife.22311.010Table 2.ITC data for SNX5 PX domain binding to truncated and mutated IncE peptides*.DOI: http://dx.doi.org/10.7554/eLife.22311.010ProteinPeptideSequenceKd (µM)△H (kcal/mol)T△S (kcal/mol)△G (kcal/mol)NSNX5 PXIncE_1PANGPAVQFFKGKNGSADQVILVTQ0.95 ± 0.07−6.9 ± 0.3−1.9 ± 0.05−8.2 ± 0.011.01 ± 0.01IncE_2 ANGPAVQFFKGKNGSADQVILVTQ1−5.0−2.6−8.10.98IncE_3 NGPAVQFFKGKNGSADQVILVTQ0.93−6.7−1.4−8.11.03IncE_4 GPAVQFFKGKNGSADQVILVTQ0.87−6.8−1.2−8.21.03IncE_5 PAVQFFKGKNGSADQVILVTQ2−5.9−1.2−8.30.99IncE_6 AVQFFKGKNGSADQVILVTQ/////IncE_7 VQFFKGKNGSADQVILVTQ2.2−6.9−1.1−7.70.99IncE_8 QFFKGKNGSADQVILVTQNo binding////IncE_9 FFKGKNGSADQVILVTQNo binding////IncE_10 FKGKNGSADQVILVTQNo binding////IncE_11 KGKNGSADQVILVTQNo binding////IncE_12 GKNGSADQVILVTQNo binding////IncE_13 KNGSADQVILVTQNo binding////IncE_14PANGPAVQFFKGKNGSADQVILVT0.72−5.1−1.6−8.41IncE_15PANGPAVQFFKGKNGSADQVILV0.97−6.5−1.3−8.20.98IncE_16PANGPAVQFFKGKNGSADQVIL1.1−5.6−1.4−8.120.99IncE_17PANGPAVQFFKGKNGSADQVI8.7−2.7−2.5−6.90.99IncE_18PANGPAVQFFKGKNGSADQVNo binding////IncE_19PANGPAVQFFKGKNGSADQNo binding////IncE_20PANGPAVQFFKGKNGSADNo binding////IncE_21PANGPAVQFFKGKNGSANo binding////IncE_22PANGPAVQFFKGKNGSNo binding////IncE_23PANGPAVQFFKGKNGNo binding////IncE_24PANGPAVQFFKGKNNo binding////IncE Q115APANGPAVAFFKGKNGSADQVILVTQ6.3−5.3−1.6−6.90.90IncE F116DPANGPAVQAFKGKNGSADQVILVTQNo bindingIncE K118APANGPAVQFFAGKNGSADQVILVTQ2.8−6.0−1.5−7.50.91IncE V127DPANGPAVQFFKGKNGSADQDILVTQNo bindingSNX5 PX L133DIncE_1PANGPAVQFFKGKNGSADQVILVTQNo bindingSNX5 PX F136AIncE_1PANGPAVQFFKGKNGSADQVILVTQNo bindingSNX5 PX E144AIncE_1PANGPAVQFFKGKNGSADQVILVTQ15−9.9−3.1−130.99*Except for IncE_1 all other peptide-binding experiments were performed only once.