(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.