To verify the crystal structure we mutated residues from both SNX5 and IncE and measured their affinities using ITC (Figure 5A and B; Table 2). At the interface between SNX5 and IncE several side chains make key contributions to peptide recognition. Because Leu133 and Phe136 residues in SNX5 are located at the core of the IncE-binding interface, and also due to the structural rearrangements these residues make on binding (see below), we reasoned that L133D and F136A mutations would inhibit the interaction. Indeed these mutants abolished association with the IncE peptide (Figure 5A). The reciprocal mutations in IncE residues F116A and V127D also abolished binding to the SNX5 PX domain (Figure 5B), and the SNX6 and SNX32 PX domains (Figure 5—figure supplement 1), demonstrating the importance of these hydrophobic and π-stacking interactions for stable complex formation. In contrast mutations predicted to disrupt an observed electrostatic contact (IncE K118A or SNX5 E144A) had little effect on binding. Thus the core hydrophobic interactions are critical for IncE binding but the peripheral electrostatic contact is not essential.10.7554/eLife.22311.015Figure 5.Mutations in the SNX5 and IncE proteins prevent complex formation in vitro.(A) ITC experiments testing the effect of SNX5 mutations on IncE binding. Both L133D and F136A mutations prevented IncE binding, but the A144A mutation had little effect. (B) ITC experiments testing the effect of IncE mutations on SNX5 binding. Both F116A and V127D blocked SNX5 interaction, while Q115A had a partial effect and K118A had no effect on association.DOI: http://dx.doi.org/10.7554/eLife.22311.01510.7554/eLife.22311.016Figure 5—figure supplement 1.SNX6 and SNX32 PX domains bind IncE at the same site as SNX5.ITC experiments testing the effect of IncE peptide mutations on binding to SNX6 and SNX32 PX domains. The IncE F116A mutation blocks interaction with both PX domains similarly to SNX5 (Figure 5B).DOI: http://dx.doi.org/10.7554/eLife.22311.016