Cryo-EM structure of the sub-50 kD PDZ-RhoGEF catalytic core-RhoA complex reveals an allosteric mechanism for GEF switch

Abstract

PDZ-RhoGEF (PRG) drives cellular processes including migration through activation of the GTPase RhoA. The GEF switch (GS) is an accessory region N-terminal to the PRG catalytic core that significantly enhances its activity against RhoA, but its mechanism is unknown. We take an integrative structural biology approach, including cryogenic electron microscopy, to deduce the GS mechanism. Enzymatic assays define interactions between the GS and PRG catalytic core that are required for enhancement of activity. While SEC-SAXS data suggest that the GS does not alter the global structure of the catalytic core-RhoA complex, HDX-MS data reveal that the GS induces widespread stabilization, particularly in the RhoA switch regions. We determined a sub-4 angstrom resolution structure of 46 kD of the complex between the catalytic core and RhoA, revealing that Switch I is more flexible without the GS. Collectively, these data suggest both direct and allosteric factors in the mechanism of the GS.