Prediction of binding sites by docking simulation based on the result from HDX-MS reveals which residues contribute to the intramolecular interaction of JRAB/MICAL-L2. (a,b) Numbers of contacts of residues in JRAB-C with JRAB-LIM (a), and in JRAB-LIM with JRAB-C (b), in the docking poses between the structural models of JRAB-C and JRAB-LIM. The residues colored in red that appeared frequently, M884, W887, Q895, and L898 in helix 2 of JRAB-C (a) and, L197, V198, and R200 in JRAB-ZF1 (b), were selected as candidates for the residues involved in the interaction between JRAB-C and JRAB-LIM. Electrostatically positive and negative regions on molecular surfaces are depicted in blue and red, respectively, and hydrophobic residues in yellow. (c) Structures of two JRAB-C double mutants (M884K/W887K and Q895K/L898K) and JRAB-LIM triple mutant (L197E/V198E/R200E), in which the side-chain conformations of the mutated residues were predicted using Scwrl 4.0. (d) HEK293 cell lysates containing GFP-tagged JRAB-C WT or mutant (M884K/W887K or Q895K/L898K) were subjected to pull-down assays using GST-JRAB-CH + LIM. The pulled-down protein was detected by WB using an anti-GFP antibody. (e) HEK293 cell lysates containing GFP-tagged JRAB-CH + LIM WT or mutant (L197E/V198E/R200E) were subjected to pull-down assays using GST-JRAB-C. The pulled-down protein was detected by WB using anti-GFP antibody. (f) Cell lysates from HEK293 cells expressing GFP-JRAB-C WT or mutant (M884K/W887K or Q895K/L898K) together with HA‐Rab13DA were immunoprecipitated (IP) with anti‐HA antibody. Each immunoprecipitate was subjected to SDS‐PAGE, followed by WB using anti‐GFP and anti‐HA antibodies. (g) Cell lysates from HEK293 cells expressing GFP-JRAB-CH + LIM WT or mutant (L197E/V198E/R200E) together with HA‐filaminA were immunoprecipitated (IP) with anti‐HA antibody. Each immunoprecipitate was detected as described in (f). (d–g) Full-length blots are presented in Supplementary Fig. S6.