The S381F substitution alters CalDAG-GEFI structure and markedly impairs its nucleotide exchange activity. (A) Bodipy fluorescence-based assay to monitor nucleotide exchange on purified Rap1B. Black arrow indicates where wild-type (green) or S381F (red) or G248W (blue) CalDAG-GEFI was added. The increase in fluorescence intensity, a measure of nucleotide exchange, over time is shown. (B) A structural model of the S381F substitution using the mutagenesis feature in pymol. This model was built on the basis of the crystal structure of CalDAG-GEFII¹⁸  and represents phenylalanine substituted for serine at position 430, the homologous position of serine 381 of CalDAG-GEFI. On the basis of this model, there are 3 residues that clash with the phenylalanine substitution at S430: leucine 263, serine 305, and leucine 309.