Figure 5. A systematic investigation of structure-specific interactors of Rab5A and Rab2A GTPases.

(A) Crystal structures of Rab5A bound to guanosine-5’-[(β,γ)-imido]triphosphate (GNP) (PDB: 1N6H) and guanosine diphosphate (GDP) (PDB: 1TU4), colored according to their secondary structural elements, are shown to underscore the conformational differences between the GNP- and GDP-bound states of Rab5A. Note that the GNP-bound structure mimics the GTP-bound form. Coil regions are highlighted in green, β-sheets in gray, and α-helices in white. The bound GNP and GDP molecules are depicted in blue, demonstrating the nucleotide interaction sites. (B, C) Barplot with numbers of significantly changing peptides (blue) and corresponding proteins (green) for Rab5A (B) or Rab2A (C) in their GTP- (left) or GDP-bound (right) forms. (D, E) Venn diagrams with the overlap of proteins identified as significantly changing upon the addition of GTP and GDP-bound forms of Rab5A (D) or Rab2A (E). (F) The plots illustrate the extent of overlap between the two nucleotide-bound forms of a bait Rab protein for the top hits in our interaction screen. In each case, the upper row shows the top 50 hits for the indicated bait protein (e.g., Rab5A-GTP) based on correlation to a sigmoidal fit of the dose-response curve (Methods); the lower row shows the results for each candidate interactor for the other form of the protein (e.g., Rab5A-GDP). Hits are arranged alphabetically; color indicates the half-maximal response concentration, and white shows non-interactors. (G–I) Venn diagrams showing interactors identified by both MitoID and LiP–MS, compared between both forms of Rab5A and Rab2A (G), GTP-bound forms (H), and GDP-bound forms (I) of the two proteins.