The Molecular Architecture of Native BBSome Obtained by an Integrated Structural Approach

Abstract

The unique membrane composition of cilia is maintained by a diffusion barrier at the transition zone that is breached when the BBSome escorts signaling receptors out of cilia. Understanding how the BBSome removes proteins from cilia has been hampered by a lack of structural information. Here, we present a nearly complete Cα model of BBSome purified from cow retina. The model is based on a single-particle cryo-electron microscopy density map at 4.9-Å resolution that was interpreted with the help of comprehensive Rosetta-based structural modeling constrained by crosslinking mass spectrometry data. We find that BBSome subunits have a very high degree of interconnectivity, explaining the obligate nature of the complex. Furthermore, like other coat adaptors, the BBSome exists in an autoinhibited state in solution and must thus undergo a conformational change upon recruitment to membranes by the small GTPase ARL6/BBS3. Our model provides the first detailed view of the machinery enabling ciliary exit.

Keywords: BBSome; cilia; trafficking.

Figure 1.. BBSome subunits and cryo-EM density map of the BBSome.

A. Domain organization of the eight BBSome subunits. The 29 domains making up the BBSome subunits are 4 β-propellers (BBS1/2/7/9), one 4-helix bundle inserted into a β-propeller (BBS1), 4 connector helices between the β-propellers and the GAE domains (some of which are predicted to form coiled coils) (BBS1/2/7/9), 4 GAE domains (BBS1/2/7/9), 3 platform domains (BBS2/7/9), 3 hairpins (BBS2/7/9), 3 helical bundles (BBS2/7/9), 3 α-solenoids (BBS4/8^TPR1-2/8^TPR3-13), 2 PH domains (BBS5), one 3-helix bundle (BBS5), and one helical micropeptide (BBS18). B. Silver-stained 4-12% SDS-PAGE gel of the BBSome purified from retinal extract. C. Density map of the BBSome at 4.9-Å resolution obtained by single-particle cryo-EM (Map 1), showing a prominent helical bundle that is located in between the base and the top hemisphere.

Figure 2.. Manual assignment of BBSome subunit domains to densities in the cryo-EM map.

A. Diagram summarizing the binary interaction studies. Interactions were identified by yeast-two hybrid (YTH, Fig. S3B), visual immunoprecipitation (VIP), and co-immunoprecipitation (co-IP) experiments. Individual datasets are depicted in Fig. S3A. Domains are boxed when interactions were assigned to specific fragment of a given subunit. Each subunit has a different color, and domains within a subunit are shown in lighter color shades from N to C termini. The same color scheme is used in all panels. B. Inter-subunit crosslinks identified by mass spectrometry mapped onto the subunits. Each subunit is drawn to the scale of its length. The numbers identify the subunit and the superscripts denote the specific domain: βprop, β-propeller; cc, coiled coil; GAE, γ-adaptin ear; pf, platform; hp, hairpin; CtH, C-terminal helix bundle; ins, insert; link, linker; PH, pleckstrin homology. C. Upper panels: The same three views presented in Fig. 1C are shown after manual segmentation. Domains are labeled as in B. Lowerpanel:The BBSome is shown segregated into its three main structural components, the top lobe consisting of BBS2 and BBS7, the corkscrew consisting of BBS4, BBS18 and the β-propeller of BBS1, and the base, which is shown as BBS8 and the assembly of BBS5, BBS9 and the linker and GAE domains of BBS1. D. Binary interactions mapped onto flattened views of the segmented map that were used to manually assign domains of BBSome subunits to specific densities in the cryo-EM map. The symbols correspond to those used in A. F. Angular distribution for BBSome projections.

Figure 3.. Rosetta-generated Cα model of the BBSome.

A. Cα models of the 24 domains from BBSome subunits that were obtained with three different Rosetta modeling protocols (CM, ab initio, and ES; see Materials and Methods for details) and could be assembled into the Cα model of the BBSome. Although the GAE domains of BBS2 and BBS7 could be modeled using co-evolutionary data (see Fig. S4), they are not shown because they could not be satisfactorily built into the cryo-EM density map. The colors and labels are as in Figure 2. B. Nearly complete Cα model of the BBSome obtained using Rosetta to assemble the 24 domains into a complex, guided by the cryo-EM density map and XLMS data (see Materials and Methods for details). The three views are the same as in Fig. 1C. The GAE domains of BBS2 and BBS7 are not included in the final Cα model but their general placement is indicated by coloring the density map. C. Magnified views of crosslink clusters in the final BBSome model. The yellow dotted lines indicate crosslinks that were satisfied by the final Rosetta molecular model. For clarity, only selected crosslinks of each cluster are shown. Depicted crosslinks are: Top Left: 9^CtH[K789]- 4^N[K116] and 9^CtH[K810]-4^N[K116]. Top Right: 5^PH1[K87]-18[K90], 18[K90]-1^GAE[K553], 18[K93]-1^GAE[K553] and 1^GAE[K553]-2^hp[K638]. Bottom Left: 9^βprop[K218]-8^N[K181]. Bottom Middle: 7^βprop[K56]-7^cc[K352], 7^βprop[K56]-2^cc[K345], 2^cc[K360]-7^cc[K359], 2^cc[K360]-7^cc[K352], 7^cc[K359]-7^cc[K352], 2^cc[K345]-7^cc[K352] and 2^cc[K345]-7^cc[K338]. Bottom Right: 2 ^βprop[K9]-1^βprop[K69], 2 ^βprop[K13]-7^hp[K658], 4^N[K20]-7^hp[K659], 4^N[K20]-7^βprop[K222], 4^N[K5]-1^βprop[K143], 1^βprop[K192]-4^N[K25].

Figure 4.. Mapping of missense pathogenic variants onto the Cα model of the BBSome.

Missense variants causing Bardet-Biedl syndrome are shown in cyan and variants causing less severe disease phenotypes in magenta. A. Variants were placed on diagrams of each BBSome subunit. RP stands for Retinitis Pigmentosa, i.e., retinal degeneration. B. All variants were mapped onto the Cα model of the BBSome to show the spatial distribution of the variants. C. Close-up views of variants present in specific domains, with BBSome subunits colored as in Figure 3.

Figure 5.. BBSome recruitment to membranes is coupled to a conformational change.

A. Placement of the crystal structure of the BBS1^βprop, which was determined in complex with ARL6^GTP (PDB id: 4V0M; Stenson et al., 2017), into the corresponding density of the 4.9-Å cryo-EM map results in a major steric clash between ARL6^GTP and BBS7 ^βprop. The crystal structures are shown in ribbon representation and the cryo-EM map as a colored segmented map. Left, overview. Right, close-up view. B. Diagram of the predicted conformational change in the BBSome induced by ARL6^GTP binding. Left panel: In solution, the BBSome exists predominantly in a closed conformation, in which BBS1^βprop is too close to the top lobe to allow binding of ARL6^GTP. Right panel: Binding of ARL6^GTP locks the membrane-bound BBSome in an open, active conformation. This conformation would allow the BBSome to interact with cargoes and/or IFT and to cross the TZ.