Rewiring protein binding specificity in paralogous DRG/DFRP complexes.

Eukaryotes have two paralogous developmentally regulated GTP-binding (DRG) proteins: DRG1 and DRG2, both of which have a conserved binding partner called DRG family regulatory protein 1 and 2 (DFRP1 and DFRP2), respectively. DFRPs are important for the function of DRGs and interact with their respective DRG via a conserved region called the DFRP domain. Despite being highly similar, DRG1 and DRG2 have strict binding specificity for their respective DFRP. Using AlphaFold generated structure models of the human DRG/DFRP complexes, we have biochemically characterized their interactions and identified interface residues involved in determining specificity. This analysis revealed that as few as five mutations in DRG1 can switch binding from DFRP1 to DFRP2. Moreover, while DFRP1 binding confers increased stability and GTPase activity to DRG1, DFRP2 binding only supports increased stability. Overall, this work provides new insight into the structural determinants responsible for the binding specificities of the DRG/DFRP complexes. [Display omitted] • DRG1 and DRG2 are paralogs that have different specificities for DFRP-binding partners • Interface residues in DRG1 facilitate the specific binding to DFRP1 • Mutation of DRG1 interface residues can switch binding specificity from DFRP1 to DFRP2 • DFRP1 binding but not DFRP2 binding can stimulate DRG1 GTPase activity Based on AlphaFold models and sequence analyses, Westrip et al. identified binding specificity determining residues in the developmentally regulated GTP-binding protein DRG1. Mutation of these residues can switch DRG1 binding from the DRG family regulatory protein 1 (DFRP1) toward DFRP2, but only DRFP1 binding enhanced the GTPase activity of DRG1. [ABSTRACT FROM AUTHOR]