ITC experiments were performed on a Microcal iTC200 instrument at 25°C. The proteins were buffer exchanged into ITC buffer (50 mM Tris (pH 8.0) and 100 mM NaCl) by gel filtration prior to ITC experiments. IncE peptides at 750 µM were titrated into 50 µM PX domain samples. The binding data was processed using ORIGIN 7.0 with a single site binding model to determine the stoichiometry (n), the equilibrium association constant Ka (1/Kd), and the enthalpy (△H). The Gibbs free energy (△G) was calculated using the equation △G = −RTIn(Ka); binding entropy (△S) was calculated by △G = △H – T△S. Three experiments were performed for each set of samples to determine the average ± standard error of the mean (SEM) for thermodynamic quantities, except for the peptide truncation experiments where only single experiments were performed. For these truncated peptide experiments, all experiments were performed using the same batch of protein to allow direct comparions to be made.