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. 2015 Feb 1;128(3):553–565. doi: 10.1242/jcs.158204

Fig. 3.

Fig. 3.

Mutational analysis confirms the putative PtdInsP-binding site on the SNX27 FERM domain. (A) The amino acids contributing to the basic patch on the F3 region of the SNX27 FERM domain (see Fig. 2B,C) were mutated to test the effect on PtdInsP binding by using ITC. The binding of SNX27 R490E, R496E, R435E/R436E/K437E (RRK-E) and wild-type (WT) SNX27 are shown in red, green, blue and black respectively. No binding signal was observed for any of the mutants, confirming the basic region on SNX27 F3 is the PtdInsP-binding site. All mutants were correctly folded as shown in supplementary material Fig. S1. (B) All SNX27 FERM domain mutants demonstrate binding to PtdIns3P with identical affinity to wild-type SNX27. (C) Mutation of the SNX27 PX domain (R196A/Y197A) abolishes PtdIns3P binding (red trace) but does not inhibit PtdIns(3,4,5)P3 binding (black trace). Refer to supplementary material Table S1 for detailed thermodynamic parameters. All experiments were performed at 25°C using 20 µM protein in the cell and 500 µM PtdInsPs injected from the syringe. Top panels show raw data and bottom panels show integrated normalized data.