Fig. 7: Clinical Rab3GAP WMS mutants have impaired activity due to disruptions at the Rab18 binding interface as revealed by in vitro GEF assays and mutation mapping.

a SDS-PAGE gel of purified Rab3GAP complex containing Rab3GAP1 T18P, Rab3GAP1 E24V or Rab3GAP2 R426C mutations stained with Coomassie Blue. 2D class averages comparing the general architecture of WT Rab3GAP to complexes containing T18P, E24V or R426C point mutations. SDS-PAGE gel were performed in biological triplicate (n = 3). b In vitro GEF assays with clinical Rab3GAP WMS mutants to detect activity towards Rab18. Nucleotide exchange was detected by measuring fluorescent decrease in reactions containing 0 nM GEF (Mock) or 300 nM GEF with 4 µM Mant-GDP loaded Rab18 and 100 µM GTPγS. Data are presented as mean with error bars showing SEM for assays performed in technical triplicate (n = 3). c Zoomed in view showing Rab3GAP WMS mutants mapped onto the cryo-EM structure of core Rab3GAP. Residues mutated in WMS are shown as sticks and the predicted position of the T18P mutation is circled in black. d Zoomed in view of the AlphaFold3 generated model showing the WMS mutants mapped to the predicted Rab18 binding interface. Residues mutated in WMS and residues predicted to interact electrostatically with WMS residues are shown as sticks and labeled. Rab3GAP1 in light blue, Rab3GAP2(1–544) in pink and Rab18 in cyan. Source data are provided as a Source Data file.