Fig. 6

Molecular basis of SH3BP5 specificity and generation of GEF-deficient mutants. a In vitro GEF assay of SH3BP5 on Switch I Rab11 mutations. Nucleotide exchange was monitored by measuring the fluorescent signal during the SH3BP5 (1–455) (300 nM) catalyzed release of Mant-GDP from 4 µM of the indicated Rab in the presence of 100 µM GTPγS. Fluorescent measurements were completed every 11 s for a total of 60 min (Excitation λ = 366 nm; Emission λ = 443 nm). Two GEF curves are shown for each condition. b Nucleotide exchange rates of Rab11A mutants plotted as a function of SH3BP5 concentration. The k_cat/K_m values for all switch I Rab11A mutations were calculated from the slope. All points have error bars, some are smaller than the size of the point. c Zoomed in view of the binding interface of SH3BP5 and Rab11. Key Rab11 residues involved in the interface with SH3BP5 are shown as sticks and labeled on the structure. Regions in SH3BP5 critical for GEF activity (LNQ52, LE250) are highlighted in red. d GEF assay of WT SH3BP5 (52 nM) and GEF-deficient mutants SH3BP5 (LNQ52AAA) and SH3BP5(LE250AK) (1000 nM), in the presence of the same concentrations of Rab11A and GTPγS as described in a. Two GEF curves are shown for each condition. e Quantification of Rab11 GEF activity for WT SH3BP5 and GEF-deficient mutations. For all panels error bars represent SD (n = 3)