Figure 3

GABARAP residues S53 and S62 are essential for Nef binding. (A) Human ATG8s sequence alignment indicates putative key residues for Nef binding specificity. Protein names of ATG8s showing Nef binding during pull-down and immunoprecipitation analysis are given in different shades of yellow, and include all members of the GABARAP subfamily. Protein names of LC3B and of the other LC3s are given in different shades of brown. Purple arrows indicate residues of GABARAP showing chemical shift changes higher than 0.05 ppm (see Fig. 2C) upon Nef titration. Residues forming HP1 and HP2 are shaded in light and dark blue, respectively. Red asterisks highlight putative key positions for determining Nef-binding specificity. The corresponding amino acids found in GABARAP and LC3B at these positions are highlighted in red. (B) Visualization of HP1 and HP2 on the surface of the GABARAP structure [PDB ID: 1KOT]. (C) Structural overlay of GABARAP and LC3B with the putative key residues necessary for Nef-binding highlighted. Cartoon representations of GABARAP (yellow) and LC3B (brown) [PDB ID: 1V49] showing their regular secondary structure elements demonstrate that S53 and F62 of GABARAP as well as the corresponding D56 and K65 of LC3B are surface exposed, and therefore are available for ligand binding. Details highlight the side chain orientations of the key residues, for this the structures have been rotated appropriately. (D) Nef-conjugated or free Sepharose beads (control) were incubated with GABARAP(S53D/F62K) or LC3B(D56S/K65F) mutants. The input, the unbound material of the flow through (U),the wash (W) fractions and the eluate (E) fractions were subjected to SDS-PAGE and visualized by CBB staining. Full-length gels are presented in Supplementary Fig. 7.