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. 2014 Dec;21(12):1035-41.
doi: 10.1038/nsmb.2920. Epub 2014 Nov 17.

Structural Basis for Membrane Targeting of the BBSome by ARL6

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Free PMC article

Structural Basis for Membrane Targeting of the BBSome by ARL6

André Mourão et al. Nat Struct Mol Biol. .
Free PMC article

Abstract

The BBSome is a coat-like ciliary trafficking complex composed of proteins mutated in Bardet-Biedl syndrome (BBS). A critical step in BBSome-mediated sorting is recruitment of the BBSome to membranes by the GTP-bound Arf-like GTPase ARL6. We have determined crystal structures of Chlamydomonas reinhardtii ARL6-GDP, ARL6-GTP and the ARL6-GTP-BBS1 complex. The structures demonstrate how ARL6-GTP binds the BBS1 β-propeller at blades 1 and 7 and explain why GTP- but not GDP-bound ARL6 can recruit the BBSome to membranes. Single point mutations in the ARL6-GTP-BBS1 interface abolish the interaction of ARL6 with the BBSome and prevent the import of BBSomes into cilia. Furthermore, we show that BBS1 with the M390R mutation, responsible for 30% of all reported BBS disease cases, fails to interact with ARL6-GTP, thus providing a molecular rationale for patient pathologies.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Structure of Chlamydomonasreinhardtii ARL6ΔN–GTP–BBS1 complex
(a)(left) Size exclusion chromatography profile of CrBBS1N (blue), CrARL6ΔN–GTP (green) and the CrARL6ΔN–GTP–CrBBS1N complex (black). The complex displays a significant shift in elution volume compared to either of the two individual proteins. (right) SDS PAGE gel of peak fractions stained with Coomassie. (b) Isothermal titration calorimetry with purified CrARL6ΔN–GTP and CrBBS1N protein reveals a 1:1 complex with a dissociation constant of 0.35±0.045μM (S.D. calculated from 3 independent experiments). (c) Crystal structure of the CrARL6ΔN–GTP–CrBBS1N complex shown in cartoon representation. The GTP molecule is shown as sticks, the Mg2+ ion as a yellow ball. N- and C-termini as well as secondary structure elements of ARL6 are indicated. The black dotted line represents an extended loop region without interpretable electron density in our structure. This extension is predicted to contain αhelical structure.(d) Perpendicular view compared to panel (c) with the seven blades of the BBS1 β-propeller labeled 1–7 and the four β-strands in blade 7 labeled A–D
Fig. 2
Fig. 2. Structural basis of the ARL6–BBS1interaction
(a) Surface rendering of the CrARL6ΔN–GTP–CrBBS1N structure with non-conserved residues colored white and conserved residues colored red. After rotation of the CrARL6 and BBS1N subunits to reveal the interaction surfaces, the positions of the highly conserved interacting residues are indicated. (b) Zoom-in on the hydrophobic part of the interface (top) and hydrophilic part of the interface (bottom) with the interacting residues displayed as sticks. (c) Capture of native BBSome from bovine retinal extracts using GST-tagged human ARL6 variants. Whereas GST-HsARL6Q73LΔN efficiently pulls down the BBSome (assessed by BBS4 immunoblotting, top), structure-based HsARL6 interface point-mutations prevent interaction with the BBSome. The membrane was post-stained with Coomassie Brilliant Blue (bottom) to reveal equal amounts of ARL6 variants in the eluates. Control capture (last lane) was performed in the absence of GST-HsARL6. (d) Pull-down of recombinantly expressed wild-type or structure-guided interface mutations of human His-BBS1N using GST-ARL6Q73L. As in panel (c), single point-mutations of conserved residues of the ARL6-BBS1N interface prevent the interaction between the two proteins as visualized by anti-His-tag antibody staining.
Fig. 3
Fig. 3. Disruption of the ARL6–BBS1 interface prevents recruitment of the BBSome to cilia
(a) Recruitment of the BBSome to cilia of clonal RPE-hTERT cell lines stably expressing either HsARL6 or HsARL6E108A with either GFP or mNeonGreen C-terminal fluorescent tags (green). Endogenous ARL6 was knocked down with ansiRNA targeting the 3’ UTR. Cells were then serum starved and immunostained for BBS5 (red) and polyglutamylated tubulin (GT335 antibody, white). (b) BBS5-positive cilia in the experiment shown in panel (a) were counted. Both untreated and a control siRNA had similar percentages of BBS5-positive cilia. The error bars represent SE between microscope fields and are technical replicates of three or more experiments.
Fig. 4
Fig. 4. Nucleotide-depended conformational changes in CrARL6
(a) Superpositioning of CrARL6ΔNin the GDP- and GTP–bound forms reveals a canonical 2 residue shift in the interswitch region. (b) Comparison of the switch regions of CrARL6ΔN–GTP in context of the CrARL6ΔN–GTP–BBS1N complex with the CrARL6ΔN–GDP structure (boxed region represents a zoom-in).
Fig 5
Fig 5. HsBBS1 M390R patient mutant does not bind HsARL6–GTP
(a) Structural mapping of M390 and E234 residues mutated in BBS onto the model of the HsARL6ΔN–GTP-HsBBS1N complex. M390 is located in blade 1 close to the ARL6-interaction interface whereas E234 is located at the top of the β-propeller far away from the ARL6-interaction interface. (b) Circular dichroism (CD) spectra of WT, E234K and M390R mutants of the HsBBS1N protein. Secondary structure content is tabulated below. (c) GST pull-down of wild-type or mutant HsBBS1N with GST-tagged HsARL6Q73LΔN. (d) Tabulation of dissociation constants (Kd) from ITC titrations of HsARL6Q73LΔN to BBS1N, wild-type, E234K and M390R. (S.D. calculated from 3 independent experiments, see Supplementary Fig. 1 for ITC curves).
Fig 6
Fig 6. Structural comparison of membrane recruitment of coating complexes by Arf, Sar and Arf-like proteins
Recruitment of Arf, Sar and ArlGTPases (shown in green) to lipid bilayers (top) requires the active GTP–bound state, which exposes an N-terminal amphipathic helix (not shown) and allow for effector-binding. The complexes of Sar1-Sec23, Arf1-γ-COP, Arf1-AP-1 and Arf1-GAT are shown in cartoon representation after structural superpositioning with the ARL6-BBS1 complex (only GTPase domains superposed). Switch regions are labeled similarly to Fig. 1. Affinities of the Arf-effector complexes are indicated below.

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