The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2

Abstract

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2^E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2^E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2^E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2^E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2^E62K retains binding to an NADPH oxidase (NOX2) subunit, p67^phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2^E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

Keywords: GTPase-activating protein (GAP); NADPH oxidase; NADPH oxidase (NOX2); RAS; Ras protein; Ras-related C3 botulinum toxin substrate 2 (RAC2); T-cell lymphoma and metastasis 1 (TIAM1); dedicator of cytokinesis (DOCK); guanine nucleotide exchange factor (GEF); immunodeficiency; molecular dynamics; molecular dynamics simulations; nucleotide exchange factor (GEF); p21-activated kinase (PAK); serine/threonine-protein kinase PAK1.