Most eukaryotic cells have a solitary primary cilium capable of sensing both internal and external stimuli (Singla and Reiter, 2006). To achieve sensitivity, cilia segregate and concentrate components of signal transduction pathways (Pala et al., 2017). The prototypical example of this concentrating effect is the crowding of rhodopsin within the elaborately modified ciliary membranes of retinal photoreceptor neurons (Sung and Chuang, 2010). Furthermore, the transport of signaling proteins in and out of the cilium allows spatial control of signal transduction, as seen in the anti-correlated movement of Patched and Smoothened during Hedgehog signaling (Rohatgi et al., 2007).

The establishment of signaling pathways within cilia relies on three processes: trafficking of proteins to cilia from the cytoplasm, selective passage through a diffusion barrier known as the transition zone at the base of the cilium (Reiter et al., 2012), and a cilium-specific internal transport mechanism known as intraflagellar transport (IFT) (Kozminski et al., 1993). The BBSome (an octameric complex of BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9, and BBS18 [Jin et al., 2010; Loktev et al., 2008]) was initially implicated in the import of transmembrane proteins into the cilium as it directly binds cytosolic ciliary targeting sequences of transmembrane proteins (Berbari et al., 2008a; Jin et al., 2010), it is enriched at the transition zone (Blacque et al., 2004; Dean et al., 2016), and migrates bidirectionally during IFT with the IFT-A and IFT-B complexes (Lechtreck et al., 2009; Liew et al., 2014; Ou et al., 2005; Williams et al., 2014). Furthermore, ciliary G-protein coupled receptors (GPCRs) including rhodopsin (Abd-El-Barr et al., 2007; Nishimura et al., 2004), somatostatin receptor 3 (SSTR3) (Berbari et al., 2008b) and neuropeptide Y receptor (Loktev and Jackson, 2013) were mislocalized in mice lacking BBSome subunits. Trafficking of non-GPCRs, including the polycystic kidney disease ion channel polycystin-1, was also affected (Su et al., 2014). However, other transmembrane proteins accumulate in BBSome-deficient cilia (Domire et al., 2011; Lechtreck et al., 2013) including those not normally destined for cilia (Datta et al., 2015). This led to a model in which the BBSome promotes retrieval and export of specific transmembrane proteins from the cilium (Nachury, 2018) and the IFT-A complex promotes entry (Mukhopadhyay et al., 2010) (Hirano et al., 2017). Studies have implicated the BBSome in the exit of phospholipase D (Lechtreck et al., 2013) and SSTR3 and Smoothened (Ye et al., 2018) from the cilium. Thus, the BBSome is a key regulator of the composition of transmembrane proteins in the ciliary membrane, and is thought to be evolutionarily related to other transmembrane protein trafficking complexes including clathrin coats and the COPI and COPII coatomers (Jin et al., 2010; van Dam et al., 2013).

Mutations in BBSome subunits are associated with Bardet-Biedl syndrome (BBS), a ciliopathy characterized by obesity, neurocognitive impairment, postaxial polydactyly, renal anomalies, and retinal dystrophy (Green et al., 1989). The disruption of the spatial organization of cilia-dependent signaling pathways may underpin many of these diverse phenotypes, including retinal degeneration (Zhang et al., 2013) and obesity (Guo and Rahmouni, 2011).

The recruitment of the BBSome to ciliary membranes (where it binds transmembrane protein substrates) is mediated by a highly specific interaction with ARL6 (also known as BBS3) (Jin et al., 2010). ARL6 is a cilium-specific (Fan et al., 2004) member of the Arf family of small GTPases, which have amphipathic N-terminal helices that associate with membranes in a GTP-dependent manner (Gillingham and Munro, 2007). ARL6 directly regulates the entry of the BBSome into cilia, as shown by an 8-fold reduction in BBSome-positive cilia following siRNA-mediated knockdown of endogenous ARL6 (Jin et al., 2010). The interaction between the BBSome and ARL6:GTP has been mapped to the N-terminal β-propeller domain of BBS1 (BBS1^βprop), and a crystal structure of this complex using recombinant proteins from Chlamydomonas reinhardtii has shown that ARL6 binds blades 1 and 7 of BBS1^βprop (Mourão et al., 2014).

Structural information for the BBSome has recently become available in the form of negative-stain reconstructions of recombinant subcomplexes (Klink et al., 2017; Ludlam et al., 2019) and a mid-resolution (4.9 Å) cryo-EM reconstruction of the complete native bovine BBSome (Chou et al., 2019). The latter study revealed the overall architecture of the BBSome with chemical crosslinking and cutting-edge integrated modeling approaches used to place individual subunits. One of the surprising revelations of this structure was that the BBSome was in a closed conformation incompatible with the BBS1^βprop:ARL6:GTP crystal structure (Mourão et al., 2014), suggesting a conformational change, representing an activation mechanism, must occur for the BBSome to bind ARL6. However, in the absence of high-resolution structures, unanswered questions remain about the exact atomic structure of the BBSome and its relationship to vesicle coat complexes, the mechanism of activation by ARL6, and the role of disease mutations in BBS.

Here, we use single-particle cryo-EM to determine structures of the native bovine BBSome complex with and without ARL6 at 3.5 Å and 3.1 Å resolution, respectively. These structures allow unambiguous subunit assignment and atomic models to be built for each of the eight BBSome subunits. The structures reveal the mechanism of ARL6-mediated activation and provide new insights into the pathogenesis of BBS-causing mutations and the evolutionary relationship between the BBSome and other transmembrane protein trafficking complexes.

Native BBSome complexes were isolated directly from bovine retinal tissue using recombinant, FLAG-tagged ARL6:GTP as bait (Jin et al., 2010). Since the BBSome interacts with only the GTP-bound form of ARL6, we used a dominant negative version of ARL6 that is deficient in GTPase activity. BBSome complexes and ARL6 were eluted from the affinity column and then purified by size-exclusion chromatography. During this step, the native BBSome complexes were recovered in different fractions from ARL6:GTP, indicating dissociation of ARL6 from the BBSome. BBSome complexes lacking ARL6 were further purified by ion exchange chromatography to yield homogenous samples suitable for structural analyses (Figure 1—figure supplement 1a). Immediately prior to vitrifying grids for cryo-EM, the BBSome samples were mixed with a 2 × molar excess of recombinant ARL6:GTP in the pursuit of reconstituting the BBSome:ARL6:GTP complex.

Three-dimensional classification of the cryo-EM data (Figure 1—figure supplement 1b–d) revealed that BBSome complexes with and without ARL6 were captured. The BBSome alone was resolved to 3.1 Å resolution and the BBSome:ARL6:GTP complex to 3.5 Å resolution (Figure 1—figure supplement 1e and Table 1). We also isolated BBSome complexes that lack BBS5 or BBS7 (Figure 1—figure supplement 1d). These rare subcomplexes (2–4% of the total dataset) may reflect native intermediates, or dissociation of the complex during purification or vitrification. Compared to the previous mid-resolution structure (Chou et al., 2019), our higher-resolution data allows atomic models to be built with sidechain accuracy including for previously unbuilt domains of BBS2 and BBS7. The higher-resolution data also revealed that the N-terminal β-propeller domains of BBS2 (BBS2^βprop) and BBS7 (BBS7^βprop) and the pleckstrin homology domains of BBS5 had been misplaced at lower resolution.

Overall architecture of the BBSome

In the absence of ARL6, the eight subunits of the BBSome are arranged in two lobes that we call the head and the body (referred to as the top and base lobes by Chou and colleagues [Chou et al., 2019]) (Figure 1). The head is formed by an asymmetric heterodimer of BBS2 and BBS7, with the other six subunits forming the body. The head and body are connected by a helical neck formed from two abutting coiled coils, one from BBS2 and the other from BBS9. BBS1^βprop occupies a special position in the BBSome, cradled loosely between BBS7 in the head and BBS4 in the body. The division of the BBSome into head and body lobes with BBS1^βprop considered separately is based on both the physical architecture and differences in dynamics. Relative to the body, the head is more flexible and less well resolved, while BBS1^βprop shows additional flexibility independent of the head movement (Figure 1—figure supplement 1g). To visualize the interlobe movement and generate high quality maps for model building, we used multibody refinement. The results show that the head adopts an ensemble of conformations with no single trajectory dominating (Video 1).

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Video 1

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Within the body, BBS9 interacts with all other subunits of the BBSome. The extensive interconnectivity may explain why the BBSome needs three dedicated chaperonin-like BBS proteins (BBS6, BBS10, and BBS12) and CCT/TRiC family chaperonins to assemble (Seo et al., 2010).

BBS1, BBS2, BBS7 and BBS9 are structural homologs

BBS2, BBS7, and BBS9 all share the same five-domain architecture with an N-terminal β-propeller (βprop) followed by a heterodimerization α-helix (hx), an immunoglobulin-like GAE domain (GAE), a mixed α/β plaform (pf), and an α-helical coiled-coil (CC) (Figure 2a). BBS1 is a shorter homolog that lacks the last two domains. The conserved domain architectures of BBS1, BBS2, BBS7 and BBS9 suggest a common evolutionary origin. Together these four structurally homologous proteins are responsible for two-thirds of the molecular mass of the BBSome (Table 2), including all of the head.

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Table 2

Protein	NCBI accession	Protein length (residues)	Molecular mass (kDa)	Total built residues (BBSome)	Total built residues (BBSome: ARL6:GTP)
BBS1	XP_010819476.1	668	72.9	486	486
BBS2	NP_001033249.1	721	79.8	659	659
BBS4	NP_001069424.1	519	58.2	386	391
BBS5	NP_001094602	341	38.8	300	300
BBS7	NP_001178275.2	715	80.4	698	706
BBS8	XP_024853996	501	56.6	475	475
BBS9	NP_001179782	887	99.1	764	764
BBS18	XP_003587939.1	69	8.1	52	52
ARL6	NP_001069250.1	186	21.1	-	167

The β-propeller domains of the four homologous BBS subunits are closely related members of the seven-bladed WD40 repeat family (Figure 2b). Each β-propeller has a ‘velcro’ closure with the N-terminal β1-strand serving as the outermost strand for the last blade (Figure 2c). Only BBS1^βprop contains a large structured insertion, with a helical region between residues 110 and 195 (Figure 2d). This insertion is one of the few regions of the BBSome for which we cannot build an accurate atomic model. Although the function of this insertion is unclear, if forms multiple chemical crosslinks with the disordered N-terminus of BBS4 (Chou et al., 2019), and has been suggested to bind PCM-1 at centriolar satellites during BBSome assembly (Chou et al., 2019; Kim et al., 2004).

BBS2^βprop is unique among the BBSome β-propeller domains, as contains two Dx[D/N]xDG-like calcium-binding loops (Rigden et al., 2011); the first in blade 4 and the second in blade 6. A calcium cation can be seen bound to both loops, coordinated by a network of acidic sidechains and the mainchain of the conserved glycine (Figure 2e). A mutation (D170N) in the first of these loops is associated with BBS (Patel et al., 2016), suggesting that calcium binding by BBS2 is required for the proper functioning of the BBSome.

The β-propeller domains of BBS1, 2, 7 and 9 are followed by an α-helix and an immunoglobulin-like β-sandwich (Figure 3a–c and Figure 3—figure supplement 1a). This β-sandwich structurally resembles GAE domains, which are found in two different types of clathrin adaptors; the adaptin subunits of clathrin adaptor protein (AP) complexes (Owen et al., 1999; Traub et al., 1999) and the monomeric GGA family of clathrin adaptor proteins (Dell'Angelica et al., 2000). However, the GAE domains of the BBSome and clathrin adaptors differ in both topology − the β4 strand participates in different β-sheets (Figure 3—figure supplement 1b–c) − and function. Whereas the GAE domains of clathrin adaptors recruit accessory proteins to clathrin by binding hydrophobic motifs within the cytosolic tails of transmembrane proteins (Brett et al., 2002; Miller et al., 2003), the BBSome GAE domains are involved in heterodimerization. BBS2^GAE dimerizes with BBS7^GAE in the head (Figure 3b), and BBS1^GAE dimerizes with BBS9^GAE in the body (Figure 3c). The dimerization interface occludes the peptide-binding site of the clathrin adaptor GAE domains (Brett et al., 2002; Jürgens et al., 2013; Miller et al., 2003) (Figure 3d). The BBSome GAE domains also show low sequence and structural similarity with one another. For example, both BBS7^GAE and BBS9^GAE have a strand insertion between the β3 and β4 strands, but this additional strand contributes to different β-sheets in the two subunits (Figure 3—figure supplement 1b). These structural differences likely prevent incorrect pairing between BBSome subunits during assembly. The α-helix that precedes the GAE domain is part of the dimerization interface and forms a short coiled-coil with the corresponding α-helix of its partner subunit (Figure 3a–c).

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In BBS2, BBS7 and BBS9, the GAE domain is followed by a domain which consists of a single β-sheet and two α-helices (Figure 3—figure supplement 1d). This domain resembles the platform domain that follows the GAE domain in the α-adaptin subunit of the clathrin AP-2 complex (Owen et al., 1999; Traub et al., 1999) but lacks the N-terminal α-helix and has an additional C-terminal β-strand (Figure 3—figure supplement 1e–f). Like the GAE and platform domains of α-adaptin, the GAE and platform domains of the BBSome subunits make extensive, hydrophobic contacts with one another (with an interface of 520–610 Ų). The relative orientation between the GAE and pf domains of BBS9 mirrors those of α-adaptin (Owen et al., 1999; Traub et al., 1999) and the modules are closely superimposable (Figure 3e). However, the same domains in BBS2 and BBS7 adopt different orientations relative to one another (Figure 3e, inset), which prevents the modules being readily superimposable with α-adaptin. Like the GAE domain, the platform domain of α-adaptin is capable of binding substrate peptides through a hydrophobic pocket (Brett et al., 2002). In the BBSome, these platform domains are solvent accessible, but are yet to be implicated in substrate recognition.

In all three subunits, the platform domain is followed by a helical C-terminal region containing a coiled-coil. The coiled-coils of BBS2 and BBS9 come together to form the neck of the BBSome (Figure 1b). The coiled-coil of BBS7 is unpaired but contacts the midpoint of the neck (Figure 3a).

Domain organization of the body

BBS4, BBS5, BBS8 and BBS18 make up the remaining third of the molecular mass of the BBSome. BBS4 and BBS8 are related proteins with tetratricopeptide repeats (TPRs) that fold into α-solenoids (Figure 4). BBS8 occupies a central region of the body whereas BBS4 runs along the side (Figure 4b). The two subunits are physically connected with the C-terminus of BBS8 binding perpendicular to the midsection of BBS4 (Figure 4c). Whereas BBS4 forms a conventional uninterrupted α-solenoid, BBS8 has an insertion between the third and fourth α-helices of its α-solenoid (residues 48–158). This insertion consists of two short α-helices and long loops that fold together into a compact domain (Figure 4b–c). The density for this region is considerably weaker than for the neighboring environment, suggesting it is flexible or capable of unfolding. TPR-containing proteins typically bind a specific linear peptide within the concave surface of the α-solenoid (Zeytuni and Zarivach, 2012). In the case of the BBS4 and BBS8, the linear peptide is BBS18, the smallest BBSome subunit (Figure 4c). By stretching between BBS4 and BBS8, BBS18 appears to stabilize their association. In absence of BBS18, BBS4 fails to incorporate into the BBSome (Loktev et al., 2008).

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BBS5, the remaining subunit, is located at the periphery of the body in extensive contact with the edge of BBS9^βprop (Figure 5a). BBS5 contains tandem pleckstrin homology (PH) domains (BBS5^N-PH and BBS5^C-PH) and an extended C-terminus that forms additional interactions with BBS9. Each PH domain consists of two curved antiparallel β-sheets forming a hydrophobic gorge capped by an amphiphilic α-helix. Despite just 25% sequence identity, the two domains share remarkable structural similarity with a root-mean-square deviation (r.m.s.d.) of 1.0 Å (Figure 5b). Many PH domains interact with the negatively charged headgroups of phosphoinositides through an electropositive binding pocket at the apex of the domain (Ferguson et al., 2000). As recombinant full-length BBS5 and BBS5^N-PH have been shown to interact with phosphatidic acid and phosphoinositides in lipid-protein overlay assays (Nachury et al., 2007), we examined whether the BBS5 PH domains had retained this pocket (Figure 5c). We also considered the noncanonical phosphoinositide binding site of the PH-family GLUE domains from ESCRT complexes (Teo et al., 2006), which are structurally similar to the BBS5 PH domains (Figure 5d). For both potential binding sites, the phosphoinositide binding sites are not conserved in either BBS5^N-PH or BBS5^C-PH. Furthermore, both pockets of BBS5^N-PH and the conventional pocket of BBS5^C-PH are occluded by elements from BBS9 (Figure 5c). The GLUE-specific phosphoinositide binding site of BBS5^C-PH is open to solvent, but lacks the basic residues required to bind phosphoinositides. We therefore conclude that if BBS5 does bind phosphoinositides in vivo, it is either through an unknown interface or after a conformational change that exposes the phosphoinositide binding sites.

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ARL6-mediated activation of the BBSome

The BBSome is recruited to ciliary membranes by membrane-associated, GTP-bound ARL6 (Jin et al., 2010). A crystal structure has shown that ARL6:GTP interacts with blades 1 and 7 of the BBS1^βprop (Mourão et al., 2014). However, this binding site is occluded in the BBSome structure, as blade 7 of BBS1 forms a continuous eight-stranded β-sheet with the corresponding blade of the adjacent BBS2^βprop (Figure 6a). The occlusion of the ARL6 binding site and the general flexibility of the head had led to suggestions that the head must open to allow ARL6 to bind (Chou et al., 2019). However, our 3.5 Å resolution structure of the BBSome:ARL6:GTP complex shows that the head remains in a closed, downward conformation even in the presence of ARL6 (Figure 6b). Rather, BBS1^βprop swivels in its cradle between BBS4 and BBS7 to accommodate ARL6 (Figure 6c and Figure 6—figure supplement 1). This swiveling action involves a rotation of approximately 25° and a movement of 13 Å away from BBS2^βprop. The N-terminal half of BBS4 shows a small 2–3 Å displacement to accommodate the movement of BBS1^βprop. However, the hx and GAE domains of BBS1 remain static due to their attachment to BBS1^βprop through a flexible linker. The swiveling of BBS1^βprop opens a central cavity in the BBSome with dimensions of 50 × 15 Å, wide enough to accommodate a polypeptide chain. This cavity is flanked by the newly exposed edges of BBS1^βprop, BBS2^β-prop and BBS7^βprop as well as BBS4 and BBS8 in the body.

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The interaction between ARL6:GTP and the BBSome-bound BBS1^βprop is similar to that seen in the crystal structure of the C. reinhardtii BBS1^βprop:ARL6:GTP ternary complex (Mourão et al., 2014). The first and last blades of BBS1^βprop interact with the switch two loop and helices α3 (residues 75–78) and α4 (residues 98–108) of the GTP-bound ARL6. Density for GTP (Figure 1—figure supplement 2c) and the ordered switch loops of ARL6 are clearly visible in our reconstruction. We also see an additional interaction between ARL6 and the loop that connects BBS7^βprop with BBS7^hx (residues 320–335) (Figure 6d). This linker is disordered in the BBSome-only structure but binds along the β-edge of the central β-sheet of ARL6. The corresponding linker in BBS2 also comes close to ARL6 (Figure 6d), although the density is insufficiently resolved to build a model of this interaction. These contacts may stabilize the BBSome:ARL6 interaction and the downward position of the head in the presence of ARL6.

Structural mapping of BBS mutations

Taking advantage of our high-quality maps in which individual sidechains are well resolved, we mapped known disease mutations in human BBSome subunits and ARL6 onto the structure of the bovine BBSome:ARL6:GTP complex (Figure 7a). Pathogenic mutations were obtained from a curated list of BBS-associated mutations (Chou et al., 2019) supplemented with ARL6 mutations from the ClinVar database (Landrum et al., 2014) (Table 3). Only non-synonymous polymorphisms annotated as pathogenic in either BBS or retinitis pigmentosa were considered. BBS1 and BBS2 are the two most commonly mutated genes in BBS (Forsythe and Beales, 1993) with the majority of mutations located in their β-propeller domains. This includes the BBS1^M390R mutation, the single most common mutation found in human BBS patients and one which is sufficient to induce BBS phenotypes including retinal degeneration and obesity in a mouse model (Davis et al., 2007). Our analysis suggests that many of the mutations within BBS1^βprop and BBS2^βprop would result in the introduction of bulky or charged residues that would disrupt hydrophobic packing and correct folding, as shown experimentally for the M390R mutation introduced into recombinant BBS1^βprop (Mourão et al., 2014). The vulnerability of BBS1^βprop and BBS2^βprop reflects their important contributions to the BBSome’s autoinhibitory and activation mechanisms. Destabilizing mutations within these domains would affect formation of the head, the positioning of BBS1^βprop, and recruitment by ARL6. We also note a cluster of mutations in BBS7 (L317V, H323R, G329V, R346Q) close to its interaction site with ARL6. In particular, H323R and G329V are within the flexible linker that only becomes ordered in the presence of ARL6 (Figure 7b). Mutations within this linker may disrupt ARL6-mediated BBSome recruitment.

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Table 3

Gene	Protein mutation	Phenotype	Reference
ARL6	T31M	BBS	(Fan et al., 2004)
ARL6	T31R	BBS	(Fan et al., 2004)
ARL6	A89V	RP	(Aldahmesh et al., 2009)
ARL6	I91T	BBS	(Chandrasekar et al., 2018)
ARL6	I94T	RP	(Khan et al., 2013)
ARL6	G169A	BBS	(Young et al., 1998)
ARL6	L170W	BBS	(Fan et al., 2004)

Most mutations outside BBS1^βprop and BBS2^βprop can be rationalized as causing misfolding of individual subunits, predominantly by affecting the packing of the hydrophobic cores. Other mutations map to the interfaces between subunits. For example, BBS4^N309K (Muller et al., 2010) maps to the interface with BBS18 (Figure 7c), BBS1^E224K (Redin et al., 2012) maps to the interface between BBS1^βprop and BBS4 (Figure 7d), and BBS2^R632P (Katsanis et al., 2001) maps to the interface between the coiled-coils of BBS2 and BBS9 in the neck (Figure 7e). These mutations may affect the proper assembly of the BBSome.

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.

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Author details

1. Sandeep K Singh

   Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

2. Miao Gui

   Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

3. Fujiet Koh

   Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, United States

   Present address

   Thermo Fisher Scientific, Eindhoven, Netherlands

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   is affiliated with Thermo Fisher Scientific. The author has no financial interests to declare

4. Matthew CJ Yip

   Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, United States

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2505-9987

5. Alan Brown

   Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   For correspondence

   alan_brown@hms.harvard.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0021-0476

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