The Structural GDP/GTP Cycle of Rab11 Reveals a Novel Interface Involved in the Dynamics of Recycling Endosomes

Abstract

The small GTP-binding protein Rab11 is an essential regulator of the dynamics of recycling endosomes. Here we report the crystallographic analysis of the GDP/GTP cycle of human Rab11a, and a structure-based mutagenesis study that identifies a novel mutant phenotype. The crystal structures show that the nucleotide-sensitive switch 1 and 2 regions differ from those of other Rab proteins. In Rab11-GDP, they contribute to a close packed symmetrical dimer, which may associate to membranes in the cell and allow Rab11 to undergo GDP/ GTP cycles without recycling to the cytosol. The structure of active Rab11 delineates a three-dimensional site that includes switch 1 and is separate from the site defined by the Rab3/Rabphilin interface. It is proposed to form a novel interface for a Rab11 partner compatible with the simultaneous binding of another partner at the Rabphilin interface. Mutation of Ser 29 to Phe in this epitope resulted in morphological modifications of the recycling compartment that are distinct from those induced by the classical dominant-negative and constitutively active Rab11 mutants. Recycling endosomes condensed in the perinuclear region where they retained recycling transferrin, and they clustered Rab11-and EEA1-positive membranes. Altogether, our study suggests that this mutation impairs a specific subset of Rab11 interactions, possibly those involved in cytoskeleton-based movements driving the slow recycling pathway.

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

“…These crystals revealed formation of a helical element (residues 70–77) within switch 2 (residues 72–82) of Rab11 that was not present in structures of Rab11 GTPγS alone 31. This region of Rab11 is located at a crystallographic contact site with a symmetry‐related PI4KIIIβ molecule.…”

“…The hydrophobic triad residues, as well as Thr67 are shown in yellow. C : Structure of GTPγS loaded Rab11 (from PDB: 10IW 31). Proteins and residues are coloured according to the scheme described in B. D,E : The 2F0‐Fc density for both PI4KIIIβ bound to GTPγS and GTPγS loaded Rab11 for the hydrophobic triad and Thr67 molecules is shown.…”

Thirty-One Flavors of Drosophila Rab Proteins

“…Although other amino acid changes have been used to produce DN and CA Rab proteins, these two types of mutation have been used extensively in other laboratories and demonstrated to be effective in many tested Rab proteins (Feng et al 1995;Press et al 1998;Dinneen and Ceresa 2004a,b;Pasqualato et al 2004). Some of the Rab proteins, including Rab18, Rab40, RabX2, RabX3, RabX6, CG9807, and CG32673, do not have the conserved T/ S or Q amino acid in the GTP-or GDP-binding domain.…”

Molecular Dissection of Rab11 Binding from Coiled-Coil Formation in the Rab11-FIP2 C-Terminal Domain

“…Although appearing as a dimer in complex with FIP2, the oligomeric state of Rab11 alone is less well defined. While it was monomeric in solution by gel filtration experiments (17), it has been observed to be dimeric crystallographically (34) and diffusion measurements on Rab11 at 13 mg/ml using NMR spectroscopy show that it is a dimer (G.D. Henry & J.D. Baleja, unpublished results).…”