FIGURE 7:

Interactions between ESMI⁶²³LL and a conserved pocket in γ1-σ1A. (A) In silico model of the interaction between ESMI⁶²³LL and γ1-σ1A. The structure of the γ1-σ1A pocket of AP-1 that interacts with ESMI⁶²³LL was modeled from the crystal structure of the corresponding pocket in the α−σ2 hemicomplex of AP-2 (2JKR) (see Materials and Methods). The names of the key pocket residues in the surface-rendered yellow representation of σ1A (black font) and gray representation of γ1 (green font) are shown as yellow and orange sticks. The residues in ESMI⁶²³LL (blue font), superimposed on the pocket, are shown in gray sticks with oxygen atoms in red and nitrogen atoms in blue. (B) Two-dimensional representation of the interaction of ESMI⁶²³LL with the γ1-σ1A pocket. The model shows the leucine residues in ESMI⁶²³LL coordinating with σ1 hydrophobic residues (⁶²Y, ⁶⁵L, ⁶⁷Phe, ⁸⁸V, and ⁹⁸V), whereas the negatively charged residue ⁶¹⁹E at −4 position coordinates with positively charged residues ¹⁵R in σ1 and ¹⁵R in γ1. (C, D) Yeast three-hybrid assay shows that interaction of CLC-2ct with γ1-σ1A is inhibited by mutations of two of the residues predicted to be involved in the interaction (⁶⁵L, ⁸⁸V). (E) Coimmunoprecipitation of ClC-2-GFP and AP-1. On immunoprecipitation with mouse γ-adaptin antibodies, the presence of both ClC-2-GFP and γ-adaptin in the precipitate is observed by Western blot.