Figure 2
From: Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization
Nef selectively binds to GABARAPs in a direct manner and contacts the canonical ligand binding site of GABARAP. (A) Nef-conjugated or free control Sepharose beads were incubated with the purified recombinant ATG8 paralogs listed. The input, the unbound material of the flow through (U), the wash (W) fractions and the eluate (E) fractions have been analyzed by SDS-PAGE and CBB staining. Full-length gels are presented in Supplementary Fig. 4. (B) Mapping the Nef binding site on GABARAP by NMR titration experiments: overlay of 2D 1H-15N-HSQC spectra of [U-15N]-GABARAP (yellow) with increasing amounts of Nef (red and purple). GABARAP residues affected upon Nef binding are labelled. For better visibility the first titration step (ratio of 1:0.5 for [GABARAP]:[Nef]) has been omitted from this figure. (C) Combined chemical shift changes Δδ(1H,15N) as a function of the GABARAP sequence. Secondary structure elements are shown on top of the diagram. Residues belonging to the hydrophobic pockets 1 and 2 are highlighted in light blue and blue, respectively. Residues for which no chemical shift changes could be determined and prolines are marked by a grey diamond. A cut-off of 0.05 ppm (continuous horizontal line) was used for significance. (D) Mapping of the Nef interaction site on a schematic representation of the secondary structure (top) and the molecular surface (bottom) of GABARAP (PDB ID: 1KOT). Residues strongly affected upon Nef binding are marked in purple, whereas non-interacting residues are shown in yellow.
