Extended Data Fig. 5: GEF activity of T4L P-Rex1 insertion variants or its domains and validation of DH-PH-DEP1 structure via mutagenesis.
From: Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism
a. GEF activity of P-Rex1T4L increases upon truncation of the C-terminal domains in a comparable pattern to wild-type P-Rex1. Activity of ΔN40P-Rex1T4L,Δ1119-1211, DH-PH-DEP1T4L, and DH-PHT4L variants monitored at 100 nM using mant-GDP activity assay. For timecourse graphs, symbols show mean and error bars show S.D. of n = 3 independent experiments conducted in duplicate. For bar graphs, symbols show rate constant (kobs) from independent experiments, bars show mean and error bars show S.D. (n = 3). * p < 0.05 versus full-lengthT4L (p = 0.0392 for DH-PH-DEP1T4L and p = 0.0141 for DH-PHT4L); ^^ p = 0.0097 versus DH-PH-DEP1T4L; repeated measures one-way ANOVA with Tukey’s multiple comparisons test. b. Coomassie-stained SDS-PAGE analysis of the indicated P-Rex1T4L variants. c. Plot of relative α-helix propensity49 of each residue in the DH domain hinge helix for P-Rex1 and P-Rex2. A loss of α-helical propensity is observed in conserved residues surrounding the hinge-point (Ile237). d. Alignment of the DH domain hinge α6-helix in the autoinhibited (closed) structure and the active (open) conformation (PDB 4YON13). Structure in ball and stick format with the Cα atoms as spheres. e-f. Coomassie-stained SDS-PAGE analysis of the indicated P-Rex1 DH-PH-DEP1 mutants. g-h. GEF activity of the indicated DH-PH-DEP1 mutants. Activity of indicated P-Rex1 variants monitored at 20 nM using mant-GDP activity assay. For timecourse graphs, symbols show mean and error bars show S.D. of n = 3 independent experiments conducted in duplicate. For bar graphs, symbols show rate constant (kobs) from independent experiments, bars show mean and error bars show S.D. (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 versus DH-PH-DEP1 (p = 0.0431 for M401A/M408A, p = 0.0063 for T240K, p = 0.0398 for T240S, p = 0.0355 for L177A/L178A and p = 0.0009 for T240K), repeated measures one-way ANOVA with Dunnett’s multiple comparisons test. Numerical data for graphs in a, g and h are available as source data.
