FIGURE 4:

Computer simulation of the complex assembly of Bnr1-Bud6-Aip5. (A) Fluorescence anisotropy binding measurements of Alexa 488-labeled Bnr1-LC (1245-1375 a.a., 30 nM) and Bnr1-C (1288-1375 a.a., 30 nM) by a serial concentration of Bud6C, respectively. The average values from three biological replicates are fitted by the Hill equation to determine the K_D. (A.U., arbitrary unit.). (B) The initial structure of Bnr1-LC. (C) The most stable complex conformation (Simulation 8 in Supplemental Figure S4) among the eight sets of Bnr1-Bud6-Aip5 MD simulations (Bnr1-LC, cyan; Aip5C, orange; Bud6^core, pink). (D) The minimal distance plot of each residue between Bnr1-LC and Aip5C(pink)/Bud6^core (blue) in the final stable complex conformation, as shown in Figure 3C. (E) The initial structure of Bnr1-C. (F) The final Bnr1-C- Bud6^core -Aip5C complex conformation after MD simulation (Bnr1-C, blue; Aip5C, orange; Bud6^core, pink). (G) The minimal distance plot of each residue between Bnr1-LC (light blue, dashed curve) and Bnr1-C (dark blue, solid curve) with Bud6^core in the final stable complex conformation. (H) The conformational distribution of Bnr1-LC/Bnr1-C as a function of the contact ratio (percentage of residues contacting with Bud6^core) and the center of mass distance to Bud6^core.