FIG. 2.
In vitro analyses of Nef-AP-2 interaction determinants. (A) Purified recombinant proteins used in this experiment include hexahistidine-tagged wild-type and DD174,175AA mutant Nef, AP-2 core tagged with GST at the C terminus of α and hexahistidine at the N terminus of β2, and the ear domain of AP4 ɛ tagged at its N terminus with GST. Binding of Nef proteins to GST-tagged AP-2 core and ɛ-ear was analyzed by GST pulldown and SDS-PAGE, followed by Coomassie blue staining (top) or immunoblotting (IB) with an antibody to Nef (bottom). The input recombinant proteins were run alongside for comparison (first four lanes). Wild-type hexahistidine-tagged Nef is visible as a ∼27-kDa band in the fifth lane. This experiment is representative of three experiments with similar results. (B) GST-ɛ-ear (control) or wild-type or DD174,175AA mutant hexahistidine-tagged Nef was covalently attached to the surface of a CM5 sensor chip. Subsequently, the surfaces were probed for binding of untagged AP-2 core by SPR. Shown are control-subtracted sensorgrams of individual injections of AP-2 core (25 μM) over surfaces containing wild-type or DD174,175AA mutant hexahistidine-tagged Nef. (Inset) Plot of varying concentrations (0.5 μM to 25 μM) of AP-2 core. (C) The interaction of hexahistidine-tagged Nef with GST-tagged AP-2 core is sensitive to ionic strength. GST-tagged AP-2 core was incubated with hexahistidine-tagged Nef in the presence of increasing NaCl concentrations (0.1 M to 1 M) during the pulldown experiment. The positions of the recombinant proteins (for simplicity, tags are not indicated) are shown on the left and those of molecular mass markers (in kilodaltons) on the right of the gel.