1. Structural Biology and Molecular Biophysics
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Structure and activation mechanism of the BBSome membrane protein trafficking complex

  1. Sandeep K Singh
  2. Miao Gui
  3. Fujiet Koh
  4. Matthew CJ Yip
  5. Alan Brown  Is a corresponding author
  1. Blavatnik Institute, Harvard Medical School, United States
Research Article
Cite this article as: eLife 2020;9:e53322 doi: 10.7554/eLife.53322
7 figures, 1 video, 4 tables, 4 data sets and 1 additional file

Figures

Figure 1 with 2 supplements
Structure of the mammalian BBSome.

(a) Two views of the cryo-EM structure of the bovine BBSome (postprocessed map contoured at a threshold of 0.015 and colored by subunit). (b) Atomic models of the eight subunits of the BBSome in the same orientations as the map in panel a. The BBSome can be conceptually divided into head and body lobes (indicated with dashed lines) with the β-propeller domain of BBS1 sandwiched between. A helical neck formed from abutting coiled coils from BBS2 and BBS9 connects the head and body of the BBSome.

Figure 1—figure supplement 1
Cryo-EM data processing.

(a) Silver-stained SDS-PAGE gel showing the purity of the bovine BBSome after purification but before addition of excess ARL6 for cryo-EM analysis. The subunits are labeled by their predicted molecular masses (given in Table 2). (b) Representative micrograph. (c) Selected 2D classes showing that the BBSome adopts a variety of orientations on the cryo-EM grid. (d) Processing strategy used to computationally separate BBSome and BBSome:ARL6:GTP complexes. (e) Fourier shell correlation (FSC) curves for the BBSome (black) and BBSome:ARL6:GTP (blue) complexes. The resolution at FSC = 0.143 is given. (f) Model-to-map FSC curves for the BBSome (black) and BBSome:ARL6:GTP (blue) complexes. The FSC curves were calculated using the unsharpened maps prior to multibody refinement. The resolution at FSC = 0.5 is shown with a dashed line. (g) Unsharpened maps colored by local resolution (before multibody refinement).

Figure 1—figure supplement 2
Data quality.

(a) Representative examples of the density for each of the eight BBSome subunits. (b) Model and density map for a region of ARL6. (c) Density for the GTP in the BBSome:ARL6:GTP complex. All maps are postprocessed following multibody refinement and contoured at a threshold between 0.015–0.02. Landmark residues are labeled.

β-propeller domains of the BBSome.

(a) Domain organization of BBS1, BBS2, BBS7, and BBS9. (b) The bovine BBSome contains four homologous β-propeller domains. The positions of the calcium cations that bind BBS2βprop are marked with a star. (c) BBS9βprop rainbow colored from N to C-terminus. The N-terminal β1-strand serves as a ‘velcro’ closure for blade 7. The individual blades are numbered. (d) BBS1βprop contains a helical insertion that likely interacts with the N-terminus of BBS4. (e) Calcium-binding loops of BBS2βprop. Residue D170 is mutated in Bardet-Biedl syndrome (Patel et al., 2016).

Figure 3 with 1 supplement
BBS1, BBS2, BBS7 and BBS9 are homologous proteins with similarities to the clathrin adaptor proteins.

(a) Location of BBS1, BBS2, BBS7, and BBS9 in the BBSome, colored except for their β-propeller domains. GAE heterodimers shown in panels c and d are boxed. In the rotated view all non-colored subunits are removed for clarity. (b) Heterodimerization of BBS2 and BBS7 involves the hx-GAE module. (c) Heterodimerization of BBS1 and BBS9. (d) Superposition of BBS9GAE with the GAE domain of AP-1 clathrin adaptor subunit γ-like 2 reveals that the heterodimerization interface with BBS1GAE would occlude the substrate binding pocket. (e) The GAE-pf module of BBS2, BBS7 and BBS9 resembles the equivalent module of the AP-2 clathrin adaptor α2-adaptin. While BBS9GAE-pf superposes closely with α2-adaptin, the GAE and pf domains of BBS2 and BBS7 (inset) adopt different orientations relative to one another.

Figure 3—figure supplement 1
Topology of the BBSome GAE and platform (pf) domains.

(a) Three-dimensional model of the BBS1GAE domain with β-strands colored. (b) Topology diagrams for the GAE domains of BBS1, BBS2, BBS7, and BBS9 following the color scheme in panel a. BBS7GAE and BBS9GAE have strand insertions (shown in gray) between the β3 and β4 strands that contribute to different β-sheets in the two subunits. (c) The topology of a GAE domain from the clathrin adaptor protein GGA3 (PDB: 1P4U). Compared to the clathrin adaptor GAE domain, the β4 strand (green) of the GAE domains of the BBSome contributes to the other β-sheet. (d) Three-dimensional model of the BBS7pf domain with secondary structure elements colored. (e) Topology diagram of BBS7pf following the color scheme in panel d. BBS2pf and BBS9pf share the same topology as BBS7pf. (f) The platform domain of the AP-2 clathrin adaptor α-appendage has an additional N-terminal α-helix and but lacks the C-terminal β-strand found in the BBSome pf domains.

BBS18 spans the α-solenoids of BBS4 and BBS8.

(a) Domain organization of BBS4, BBS8 and BBS18. BBS18 is 69 residues long and does not form a globular domain. BBS8 has an insertion (ins.) between tetratricopeptide repeats 2 and 3. (b) Location of BBS4, BBS8 and BBS18 in the BBSome. (c) Two views of the BBS4-BBS8-BBS18 subcomplex. BBS8 binds perpendicular to BBS4. The insertion in BBS8 forms a globular domain made from long loops and short α-helices. BBS18 binds the concave surfaces of the BBS8 and the N-terminal half of BBS4.

BBS5.

(a) Position of BBS5 at the periphery of the BBSome body. BBS5 has tandem pleckstrin homology domains (BBS5N-PH and BBS5C-PH) and an extended C-terminus (BBS5C-term). (b) BBS5N-PH and BBS5C-PH superpose with a root-mean-square deviation (r.m.s.d.) of 0.97 Å. (c) Potential phosphoinositide binding sites were determined from crystal structures of pleckstrin homology domains in complex with inositol-(1,3,4,5)-tetrakisphosphate (PDB: 1FAO) (Ferguson et al., 2000) or sulfate ions (PDB: 2CAY) (Teo et al., 2006). Three of the four potential binding sites in BBS5 are occluded by other BBSome subunits (blue arrows). The fourth (green arrow) is accessible but not conserved. (d) Superposition of BBS5C-PH with the GLUE domain of Vps36, a component of the ESCRT-II complex (Teo et al., 2006). Vps36GLUE has a non-canonical phosphoinositide binding site (identified based on the binding site of a sulfate ion).

Figure 6 with 2 supplements
Mechanism of BBSome activation by ARL6.

(a) In the BBSome-only state, BBS1βprop and BBS2βprop bind edge-to-edge with hydrogen bonding between their β1 strands generating a continuous eight-stranded β-sheet. (b) Cryo-EM structure of the BBSome:ARL6:GTP complex (postprocessed map contoured at a threshold of 0.015 and colored by subunit). ARL6 interacts with BBS7βprop and BBS1βprop, which is in a rotated state compared to in the BBSome-only structure (Figure 1). (c) Rotation of BBS1βprop in the ARL6-bound state breaks the interaction with BBS2βprop and opens a central cavity in the BBSome. The region highlighted in panel d is boxed. (d) Details of the interaction between ARL6:GTP, BBS2, and BBS7. A loop of BBS7 that is disordered in the BBSome-only state forms a β-addition with the central β-sheet of ARL6. Regions of BBS2 and BBS7 that are not fully resolved in the cryo-EM density are shown as dashed lines.

Figure 6—figure supplement 1
Rotation of BBS1βprop upon ARL6 binding.

(a) Atomic model of the BBSome showing the positions of BBS1 in the unbound (gray) and ARL6-bound states (blue). (b) Two orthogonal views showing interatomic vectors calculated between Cα atoms of the BBSome. Each vector is colored by subunit. Only BBS1βprop shows substantial movement in the ARL6 bound state. The direction of movement is indicated by an arrow.

Figure 6—figure supplement 2
Model for the assembly of the BBSome at ciliary membranes.

(a) ARL6 determines the orientation of the BBSome at membrane. The relatively flat surface of the BBSome suggests that it runs parallel to the membrane with the opposite side interacting with the IFT complexes. (b) The BBSome as viewed from the arrow in panel a. The three subunits (BBS1, BBS2, and BBS9) that interact experimentally with IFT38 (Nozaki et al., 2019) are colored. The three domains come together at the base of the neck where the coiled-coils of BBS2 and BBS9 meet the GAE dimerization domains of BBS1 and BBS9 (circled).

Structural insights into Bardet-Biedl syndrome.

(a) Known disease-causing mutations mapped onto the model of the BBSome:ARL6:GTP complex. Each sphere, colored by subunit, represents a missense mutation associated with either BBS or retinitis pigmentosa. (b) Mutations in BBS7 close to the binding site with ARL6. (c–e), BBS mutations that could disrupt subunit association and therefore proper BBSome assembly. Panels b-e show the postprocessed map contoured at a threshold between 0.015–0.02 and colored by subunit.

Videos

Video 1
Dynamics of the BBSome:ARL6:GTP complex represented by the first three principal motions from multibody refinement.

Two orthogonal views are shown for each motion.

Tables

Table 1
Cryo-EM data collection, refinement and validation statistics.
BBSome
(EMD-21144)
(PDB 6VBU)
BBSome:ARL6:GTP
(EMD-21145)
(PDB 6VBV)
Data collection and processing
Magnification81,00081,000
Voltage (kV)300300
Electron exposure (e–/Å2)5656
Defocus range (μm)−1.1 to −2.4−1.1 to −2.4
Pixel size (Å)1.061.06
Symmetry imposedC1C1
Final particle images (no.)152,94275,201
Map resolution (Å)
FSC threshold
3.1
0.143
3.5
0.143
Refinement
Resolution limit set in refinement (Å)3.13.5
Map sharpening B factor (Å2)−45.9−43.6
Model composition
Non-hydrogen atoms
Protein residues
Ligands

30,209
3820
2 Ca2+

31,676
4000
2 Ca2+; 1 GTP
B factors (Å2)
Protein
Ligand

60.8
74.0

53.9
82.3
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)

0.005
0.68

0.004
0.71
Validation
MolProbity score
Clashscore
Poor rotamers (%)

2.02
12.0
0.2

2.07
12.7
0.7
Ramachandran plot
Favored (%)
Allowed (%)
Disallowed (%)

93.5
6.5
0.0

92.7
7.2
0.1
Table 2
Proteins present in the BBSome or BBSome:ARL6:GTP complexes.
ProteinNCBI accessionProtein length (residues)Molecular mass (kDa)Total built residues (BBSome)Total built residues (BBSome:
ARL6:GTP)
BBS1XP_010819476.166872.9486486
BBS2NP_001033249.172179.8659659
BBS4NP_001069424.151958.2386391
BBS5NP_00109460234138.8300300
BBS7NP_001178275.271580.4698706
BBS8XP_02485399650156.6475475
BBS9NP_00117978288799.1764764
BBS18XP_003587939.1698.15252
ARL6NP_001069250.118621.1-167
Table 3
Mutations in ARL6 associated with BBS or retinitis pigmentosa (RP) that are mapped onto the structure in Figure 7a.

The mapped disease-associated mutations in core BBSome subunits are provided as a supplemental table in Chou et al. (2019).

GeneProtein mutationPhenotypeReference
ARL6T31MBBS(Fan et al., 2004)
ARL6T31RBBS(Fan et al., 2004)
ARL6A89VRP(Aldahmesh et al., 2009)
ARL6I91TBBS(Chandrasekar et al., 2018)
ARL6I94TRP(Khan et al., 2013)
ARL6G169ABBS(Young et al., 1998)
ARL6L170WBBS(Fan et al., 2004)
Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Gene (Bos taurus)ARL6. NCBI Gene ID: 519014IDT-Codon optimized
Biological SampleBovine dark-adapted retinasW L Lawson company (NE, USA)-
Strain, strain background Escherichia coli cellsBL21(DE3)Novagen69450–4Chemically Competent cells
Affinity resinAnti-Flag M2SigmaA2220
Chemical compoundGTPSigmaG8877
Cryo gridsQUANTIFOIL R 1.2/1.3Electron Microscopy SciencesQ4100AR1.3
Commercial assay or kitGibsons AssemblyInvitrogenA14606
Sequence based reagentsArl6_dN16 FwdThis paperPCR primersGAAGTTCATGTGCTGTGTTTGG
Sequence based reagentsArl6_dN16 RevThis paperPCR primersACTCCCACCCCCTTTATCATC
Sequence based reagentsArl6_addHis_FwdThis paperPCR primersTG GAA GTT CTG TTC CAG GGG CCC GATTACAAGGACGATGATGATAAAG
Sequence based reagentsArl6_addHis_RevThis paperPCR primersGAATTCTCGAGCGGCCGCCCTTATGTCTTCACCGACTGAATC
Software, algorithmserialEMdoi:10.1038/s41592-019-0396-9RRID:SCR_017293https://bio3d.colorado.edu/SerialEM
Software, algorithmMotionCor2 v.1.2.1doi:10.1038/nmeth.4193RRID:SCR_016499
Software, algorithmCTFFIND v.4.1.13doi:10.1016/j.jsb.2015.08.008RRID:SCR_016732https://cistem.org/ctffind4
Software, algorithmRELION v.3.0.4doi:10.7554/eLife.42166RRID:SCR_016274https://www3.mrc-lmb.cam.ac.uk/relion/index.php/Download_%26_install
Software, algorithmCoot v. 0.9-predoi:10.1107/S0907444904019158RRID:SCR_014222https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
Software, algorithmPhenix.real_space_refinedoi:10.1107/S2059798318006551RRID:SCR_014224https://www.phenix-online.org/
Software, algorithmUCSF Chimera v1.13.1doi:10.1002/jcc.20084RRID:SCR_004097http://plato.cgl.ucsf.edu/chimera/
Software, algorithmUCSF ChimeraX v.0.9doi:10.1002/pro.3235RRID:SCR_015872https://www.cgl.ucsf.edu/chimerax/
Software, algorithmPyMOL v2.3.2PyMOL Molecular Graphics System, Schrödinger, LLCRRID:SCR_000305http://www.pymol.org/
Software, algorithmcrYOLOdoi:10.1038/s42003-019-0437-z-http://sphire.mpg.de/wiki/doku.php?id=pipeline:window:cryolo
Software, algorithmResMapdoi:10.1038/nmeth.2727-http://resmap.sourceforge.net/
Software, algorithmI-TASSERdoi:10.1186/1471-2105-9-40RRID:SCR_014627https://zhanglab.ccmb.med.umich.edu/I-TASSER
Software, algorithmMolProbity v.4.3.1doi:10.1107/S0907444909042073RRID:SCR_014226http://molprobity.biochem.duke.edu
Software, algorithmSBGriddoi: 10.7554/eLife.01456RRID:SCR_003511https://sbgrid.org/

Data availability

The EM density map for the BBSome has been deposited under accession code EMD-21144 and the EM density map for the BBSome:ARL6:GTP complex has been deposited under accession code EMD-21145. Masks and maps from multibody refinement are included as additional maps in these depositions. The corresponding atomic models have been deposited under accession codes 6VBU and 6VBV.

The following data sets were generated
  1. 1
    Electron Microscopy Data Bank
    1. SK Singh
    2. M Gui
    3. F Koh
    4. MCJ Yip
    5. A Brown
    (2020)
    ID EMD-21144. Structure of the bovine BBSome (map).
  2. 2
    RCSB Protein Data Bank
    1. SK Singh
    2. M Gui
    3. F Koh
    4. MCJ Yip
    5. A Brown
    (2020)
    ID 6VBU. Structure of the bovine BBSome (model).
  3. 3
    Electron Microscopy Data Bank
    1. SK Singh
    2. M Gui
    3. F Koh
    4. MCJ Yip
    5. A Brown
    (2020)
    ID EMD-21145. Structure of the bovine BBSome:ARL6:GTP complex (map).
  4. 4
    RCSB Protein Data Bank
    1. SK Singh
    2. M Gui
    3. F Koh
    4. MCJ Yip
    5. A Brown
    (2020)
    ID 6VBV. Structure of the bovine BBSome:ARL6:GTP complex (model).

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