Fig. 7
Mutagenesis of canonical and noncanonical sites in PX domain proteins. a Selected bacterially expressed and purified PX domains and their mutants with GST tags removed were incubated with artificial liposomes as indicated. Liposomes included POPC/POPE (PC/PE) as a negative control, Folch I as an indicator of broad membrane-binding activity, or PC/PE liposomes containing the specific lipids PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, PtdIns(3,4,5)P2, or PS. Samples were subjected to ultracentrifugation followed by SDS-PAGE and Coomassie staining of the unbound supernatant (S) and bound pellet (P) fractions. b Water-soluble PtdIns3P and PtdIns(3,4)P2 headgroup analogues (500 μM) were titrated into the SGK3 PX domain (20 μM) and measured by ITC. To test for competition between the two lipids, the SGK3 PX domain was pre-incubated with the indicated lipid before titration with the other. Top panels show the raw data and bottom panels represent the integrated and normalized data fit with a 1:1 binding model. The binding affinities (Kd) are provided in Supplementary Table 1
