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. 2006 Oct 17;103(42):15416-21.
doi: 10.1073/pnas.0605357103. Epub 2006 Oct 9.

Structural basis for Rab11-dependent membrane recruitment of a family of Rab11-interacting protein 3 (FIP3)/Arfophilin-1

Tomoo Shiba  1 , Hiroshi Koga, Hye-Won Shin, Masato Kawasaki, Ryuichi Kato, Kazuhisa Nakayama, Soichi Wakatsuki
Affiliations

Structural basis for Rab11-dependent membrane recruitment of a family of Rab11-interacting protein 3 (FIP3)/Arfophilin-1

Tomoo Shiba et al. Proc Natl Acad Sci U S A. .

Abstract

Family of Rab11-interacting protein (FIP)3/Arfophlin-1 and FIP4/Arfophilin-2 are dual effectors for Rab11 and ADP ribosylation factor (ARF)5/ARF6, which are involved in membrane delivery from recycling endosomes to the plasma membrane during cytokinesis. Here, we define the distinct C-terminal binding regions of FIP3 and FIP4 for Rab11 and ARF5/ARF6. Furthermore, we determined the crystal structure of Rab11 in complex with the Rab11-binding domain (RBD) of FIP3. The long amphiphilic alpha-helix of FIP3-RBD forms a parallel coiled-coil homodimer, with two symmetric interfaces with two Rab11 molecules. The hydrophobic side of the RBD helix is involved in homodimerization and mediates the interaction with the Rab11 switch 1 region, whereas the opposite hydrophilic side interacts with the Rab11 switch 2 and is the major factor contributing to the binding specificity. The bivalent interaction of FIP3 with Rab11 at the C terminus allows FIP3 to coordinately function with other binding partners, including ARFs.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Delineation of the regions of FIP3 and FIP4 essential for their interactions with Rab11. (Upper) Extracts of HeLa cells expressing HA-tagged Rab11(Q70L) were pulled down with GST or FIP3 (a) or FIP4 (b) GST fusion proteins, as indicated and processed for immunoblot analysis to detect bound HA-Rab11(Q71L). (Lower) The blots were then stained with Coomassie brilliant blue (CBB).
Fig. 2.
Fig. 2.
Specific interactions of FIP3 and FIP4 with ARF5/6 and delineation of the FIP ABD. (a) Extracts of HeLa cells expressing HA-tagged ARF1(Q71L) (Left), ARF5(Q71L) (Center) or ARF6(Q67L) (Right) were pulled down with GST or FIP3 (residues 607–756), FIP4 (residues 484–637), or GGA1-GAT fused to GST. (Upper) The extracts were then processed for immunoblot analysis to detect bound ARF-HA. (Lower) The blots were then stained with CBB. (b and c) Extracts of HeLa cells expressing HA-ARF5(Q71L) were pulled down with GST or FIP3 (b) or FIP4 (c) fragments fused to GST as indicated. (Upper) The extracts were then processed for immunoblot analysis to detect bound HA-ARF5(Q71L). (Lower) The blots were then stained with CBB.
Fig. 3.
Fig. 3.
Structure of the Rab11–FIP3-RBD complex. (a) Ribbon stereodiagram of the Rab11–FIP3-RBD complex (side view). The asymmetric unit contains two Rab11 (molecules A and B) and two FIP3-RBD (molecules C and D) molecules. The switch 1, switch 2, and interswitch regions of Rab11 are highlighted in green, red, and blue, respectively. GTP and Mg2+ ions are shown as ball-and-stick models. Encircled N and C stand for N and C termini, respectively. (b) Top view of a. (c) Sequence alignment of the RBD of the human FIP family proteins. Red, residues identical among all of the members; green, residues conserved among at least four members; blue, residues conserved between FIP3 and FIP4. The secondary structures of FIP3-RBD are schematically shown at the bottom. The residues involved in dimerization are marked with a + symbol. Orange (molecule C) and purple (molecule D) asterisks above the sequences stand for the residues involved in the interaction with Rab11 (molecule A).
Fig. 4.
Fig. 4.
Superposition of Rab11 in different forms. The switch 1 and switch 2 regions are shown. The side chains of Arg-72 and Arg-74 in the switch 2 region are indicated by ball-and-stick models. GTP/GDP molecules also are presented as ball-and-stick models. The GDP-bound form (PDB ID code 1OIV) is blue, the GTP-bound free form (PDB ID code 1OIW) is green, and molecule A GTP-bound Rab11 in complex with FIP3-RBD is red, whereas molecule B is orange.
Fig. 5.
Fig. 5.
Interface between Rab11 and FIP3-RBD. (a) The Rab11 and FIP3-RBD interface in an “open book” representation. (Left) Rab11 (molecule A) is shown as a ribbon diagram. (Right) The FIP3-RBD dimer. The color scheme is the same as that of Fig. 3b. The residues involved in the interactions are displayed as ball-and-stick models. (b) Molecular surface representation of a. The surfaces are colored according to the electrostatic surface potential (blue, positive; red, negative; scale, −10 to +10 kTe−1). (c) The hydrophobicity is green and is shown in the same view as a.
Fig. 6.
Fig. 6.
Interaction between Rab11 and FIP3-RBD. Shown are close-up views of the switch 1 (a), switch 2 (b), and interswitch (c) regions of Rab11 in complex with FIP3-RBD. The color scheme is the same as that of Fig. 3b. Residues involved in the interaction are labeled and indicated by ball-and-stick models. The cyan dashed lines indicate hydrogen bonds or electrostatic interactions.
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