CYRI-A is similar to CYRI-B in amino acid sequence and RAC1 binding. (A) The molecular structure of CYRI-A as predicted by I-TASSER. The protein is composed of one N-terminal amphipathic α-helix connected to a domain of unknown function (DUF1394) via a flexible linker. Numbers represent the amino acid position. (B) Multiple sequence alignment using Clustal Omega of mouse CYRI-A and CYRI-B shows 80% sequence identity. Black, identical residues; gray, similar residues; red, Glycine (G2) putative myristoylation site; green, two conserved arginine residues (RR) mediating active RAC1 binding. (C) Western blots showing GST-Trap pulldown assay of GST or GST-RAC1 (WT or Q61L) with lysate from COS-7 cells expressing GFP (negative control), eCFP-CRIB-PBD (positive control), or GFP-CYRI-A (29–319; RBD). Probed with anti-GFP and anti-GST. Black arrowhead denotes GFP-CYRIA (29–319) and GST-RAC1 Q61L interaction. (D) Quantification of C from at least three independent experiments. Signals were normalized to the input. Mean ± SEM. Black arrowhead points to CYRI-A RBD and active RAC1 interaction. (E) Western blot showing MBP-Trap pulldown assay between MBP-CYRI-A on MBP beads interacting with GST-RAC1 WT, Q61L, or T17N (constitutively inactive). (F) Quantification of E from three independent experiments. Signals were normalized to MBP. Mean ± SD. Statistical analysis using one-way ANOVA with Tukey’s multiple comparisons. ns, P > 0.05. (G) Steady-state SPR binding curve between purified MBP-tagged full-length CYRI-A (magenta) or CYRI-B (cyan) and increasing concentrations of untagged full-length RAC1 Q61L with curve fit assuming a 1:1 binding model. K_d = 1.3 × 10⁻⁵ (CYRI-A::RAC1 Q61L) and 3.8 × 10⁻⁵ (CYRI-B::RAC1 Q61L). Mean ± SD. Data from at least three independent experiments.