title: Structural basis for Rab11-mediated recruitment of FIP3 to recycling endosomes.
abstract: The Rab11 GTPase regulates recycling of internalized plasma membrane receptors and is essential for completion of cytokinesis. A family of Rab11 interacting proteins (FIPs) that conserve a C-terminal Rab-binding domain (RBD) selectively recognize the active form of Rab11. Normal completion of cytokinesis requires a complex between Rab11 and FIP3. Here, we report the crystal structure and mutational analysis of a heterotetrameric complex between constitutively active Rab11 and a FIP3 construct that includes the RBD. Two Rab11 molecules bind to dyad symmetric sites at the C terminus of FIP3, which forms a non-canonical coiled-coiled dimer with a flared C terminus and hook region. The RBD overlaps with the coiled coil and extends through the C-terminal hook. Although FIP3 engages the switch and interswitch regions of Rab11, the mode of interaction differs significantly from that of other Rab-effector complexes. In particular, the switch II region undergoes a large structural rearrangement from an ordered but non-complementary active conformation to a remodeled conformation that facilitates the interaction with FIP3. Finally, we provide evidence that FIP3 can form homo-oligomers in cells, and that a critical determinant of Rab11 binding in vitro is necessary for FIP3 recruitment to recycling endosomes during cytokinesis.
authors.affiliation: Program in Molecular Medicine and Department of Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Sudharshan
authors.initials: S
authors.lastname: Eathiraj
authors.forename: Ashwini
authors.initials: A
authors.lastname: Mishra
authors.forename: Rytis
authors.initials: R
authors.lastname: Prekeris
authors.forename: David G
authors.initials: DG
authors.lastname: Lambright
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020691:rab GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D000099193:rab11 GTP-Binding Proteins
chemical_list: D049452:Green Fluorescent Proteins; D051550:I-kappa B Kinase; D011993:Recombinant Fusion Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C491731:IKBKG protein, human