Structural basis for recruitment of RILP by small GTPase Rab7

Abstract

Rab7 regulates vesicle traffic from early to late endosomes, and from late endosomes to lysosomes. The crystal structure of Rab7-GTP in complex with the Rab7 binding domain of RILP reveals that Rab7 interacts with RILP specifically via two distinct areas, with the first one involving the switch and interswitch regions and the second one consisting of RabSF1 and RabSF4. Disruption of these interactions by mutations abrogates late endosomal/lysosomal targeting of Rab7 and RILP. The Rab7 binding domain of RILP forms a coiled-coil homodimer with two symmetric surfaces to interact with two separate Rab7-GTP molecules, forming a dyad configuration of Rab7-RILP(2)-Rab7. Mutations that disrupt RILP dimerization also abolish its interactions with Rab7-GTP and late endosomal/lysosomal targeting, suggesting that the dimeric form of RILP is a functional unit. Structural comparison suggests that the combined use of RabSF1 and RabSF4 with the switch regions may be a general mode of action for most Rab proteins in regulating membrane trafficking.

Figure 1

Structure of the Rab7–RILPe complex. (A) Stereo view of a representative portion of the F_o−F_c electron density map (contoured at 1.6σ) covering residues 256–263 of helix α1 in the Rab7 binding domain of RILP. The map was calculated with phase from Rab7 molecule only. (B) Ribbon diagram of the Rab7–RILPe complex. Two Rab7 molecules a and c are colored light green whereas two RILPe molecules b and d are red and dark green, respectively. The switch I, interswitch and switch II regions are colored cyan, royal blue and magenta, respectively. GTP molecule is shown in stick model and Mg²⁺ as cyan gray sphere. Residues 175–185 containing RabSF4 of Rab7 are shown in orange. (C) Top view of the Rab7–RILPe complex. The molecules are rotated 90° along a horizontal axis relative to the view in (B). (D) Comparison of Rab7-GTP in a free form and in complex with RILPe. Free Rab7-GTP (left panel) and that in complex with RILPe (right panel) are colored white and light green, respectively. Helix α5 of Rab7 is highlighted in red. The coloring scheme for the switch regions, GTP, Mg²⁺ and residues 175–185 of Rab7 in complex with RILPe is as in (B). Figures 1, 2 and 6 were generated using Molscript (Kraulis, 1991).

Figure 2

RILP–RILP and Rab7–RILP interfaces. (A) Stereo diagram of the first Rab7–RILP interface between the switch and interswitch regions of Rab7 and RILPe. The coloring scheme for the switch and interswitch, and RILPe molecules is as in Figure 1B, and the rest of Rab7 is yellow. (B) Stereo diagram of the second Rab7–RILP interface between the N- and C-terminal regions of Rab7 and RILPe. The N- and C-terminal regions of Rab7 are blue and magenta, respectively, with the rest of Rab7 in yellow. The coloring scheme for RILPe is as in (A). (C) The RILP–RILP interface showing the interactions of two long helices α1 from chain b (red) and d (dark green). For simplicity, only pairwise located residues involved in dimer interface are shown in stick model. (D) The RILP–RILP interface showing the interactions of the long helix α1 from chain b and the short helix α2 from chain d. The coloring scheme is as in (C). All residues involved in the interactions are shown in stick model.

Figure 3

Sequence alignment of Rab GTPases, and the Rab7 binding domain of RILP with RLPs. (A) Sequence alignment of human Rab7, yeast Ypt7p, human Rab34, human Rab3a and human Rab5c. The secondary structures of human Rab7 in complex with RILPe are shown at top. Switch and interswitch regions are marked with blue and green lines, respectively. RabSF motifs are marked with red lines. Mutated residues involved in interaction with RILP are marked with #. (B) Sequence alignment of human RILP, mouse RLP1 and human RLP2. The secondary structures of RILPe are shown at top. Mutated residues involved in RILP dimerization are marked with ^* and those involved in interactions with Rab7 are marked with •. Residues showed in Figure 2C in positions a and d of the coiled-coil helix α1 are marked with black and red triangles, respectively.

Figure 4

Effects of mutations on RILP dimerization and Rab7–RILP interaction. (A) RILP forms homodimer via self-interaction in a manner that is dependent on residues (241–260) in its coiled-coil region. Wild type (WT) refers to full-length RILP. (B) Effects of Ala point mutations of RILP on its dimerization. L287A serves as a positive control. (C) Effects of truncation or Ala point mutations in Rab7Q67L on its interaction with RILP. Rab7Q67L and E185A mutant serve as positive controls. Rab7ΔN contains residues 11–207, Rab7ΔC1 contains residues 1–176 and Rab7ΔC2 contains residues 1–185. (D) Effects of mutations of RILP on its interaction with Rab7Q67L. Wild-type (WT) RILP and RILPe serve as positive controls.

Figure 5

Effects of mutations of Rab7 and RILP on their cellular localization and membrane recruitment. (A) Representative site-directed mutants (panels e, g, i and k) or a truncated form (Rab7ΔN; panel c) of Rab7Q67L defective in interaction with RILP are mistargeted to the cytosol (and nucleus for F45A) (as revealed by GFP attached to the N-terminus of these proteins) and are not detected in the clustered lysosomes marked by coexpressed RILP (panels d, f, h, j and l as revealed by Myc tag). EGFP-Rab7Q67L (panel a) and Myc-RILP (panel b) serve as the positive control. Bar, 20 μm. (B) Representative mutants of RILP (panels c, e, g, i and k, revealed by Myc tag) defective in interaction with Rab7 are mistargeted to the cytosol and did not associate with punctuate late endosomes/lysosomes marked by coexpressed EGFP-Rab7Q67L (panels b, d, f, h, j and l, viewed by GFP). The fragment encompassing residues 241–310 of RILPe is peripheral distributed, but still can be efficiently recruited to the punctuate structures marked by EGFP-Rab7Q67L (panels a and b). Bar, 20 μm.

Figure 6

Structural comparison of the complexes of Rab3A–Rabphilin3A, Rab5–Rabaptin5, Rab7–RILP and Arl1–GRIP. The regions of GTPases involved in interactions with their effectors are highlighted in magenta. The N- and C-termini of GTPases and their effectors are labeled.