Supplementary Figure 3: The RD functions concomitant nucleotide exchange by the armadillo domain

a. Stimulation of nucleotide release by Fes1 and ∆RD. Preformed complexes of Ssa1 (0.5 µM) and MABA-ADP (0.5 µM) were rapidly mixed with 1 mM ATP in the absence or presence of Fes1 or ∆RD (2 µM). Exemplary traces of three independent experiments are shown. b and c. Dissociation rate constants of complexes of Ssa1 and a fluorescently labeled peptide in the presence of increasing concentrations of Fes1 in the absence of added nucleotides (b) and the presence of ATP (c). Ssa1 (1 µM) with bound ADP was pre-incubated for 30 min with the dansylated NRLLLTG peptide (1 µM) and then mixed with Fes1 at the indicated concentration (concentration after 1:1 mixing) in the absence of added nucleotide or in the presence of 1 mM ATP. Differences in (b) are not statistically significant (Sidak’s multiple comparison test). Data from three independent experiments are presented with their mean values. d. ATP binding to Ssa1 is accelerated by Fes1. Observed association rates of MABA-ATP to Ssa1 in the absence and presence of Fes1. MABA-ATP (4 and 8 µM; indicated are final concentration) was rapidly mixed 1:1 with nucleotide-free Ssa1 (1 µM) in the absence and presence of Fes1 (1 µM). ATP associates with Ssa1 with biexponential kinetics corresponding to the initial encounter complex (concentration dependent rate) and a subsequent conformational change (concentration independent) that leads to opening of the substrate binding domain and substrate release. Data from three independent experiments are presented with their mean values. ANOVA with Sidak's multiple comparisons test was performed; **** p<0.0001.