Abstract
Bardet-Biedl syndrome (BBS) is a genetic disorder characterized by malfunctions in primary cilia resulting from mutations that disrupt the function of the BBSome, an 8-subunit complex that plays an important role in protein transport in primary cilia. To better understand the molecular basis of BBS, here we used an integrative structural modeling approach consisting of EM and chemical cross-linking coupled with MS analyses, to analyze the structure of a BBSome 2-7-9 subcomplex consisting of three homologous BBS proteins, BBS2, BBS7, and BBS9. The resulting molecular model revealed an overall structure that resembles a flattened triangle. We found that within this structure, BBS2 and BBS7 form a tight dimer through a coiled-coil interaction and that BBS9 associates with the dimer via an interaction with the α-helical domain of BBS2. Interestingly, a BBS-associated mutation of BBS2 (R632P) is located in its α-helical domain at the interface between BBS2 and BBS9, and binding experiments indicated that this mutation disrupts the BBS2-BBS9 interaction. This finding suggests that BBSome assembly is disrupted by the R632P substitution, providing molecular insights that may explain the etiology of BBS in individuals harboring this mutation.
Highlights
Bardet-Biedl syndrome (BBS) is a genetic disorder characterized by malfunctions in primary cilia resulting from mutations that disrupt the function of the BBSome, an 8-subunit complex that plays an important role in protein transport in primary cilia
Transport of these cargos occurs through association of the BBSome with the intraflagellar transport (IFT)-B complex via an interaction with a linker protein LZTFL1 that binds to IFT27 in the IFT-B complex [15, 16, 20]
The subcomplex between BBS2, BBS7, and BBS9 is an important early intermediate in assembly of the BBSome [23]. We purified this BBS2-7-9 subcomplex for structural studies by co-expressing affinity-tagged versions of each subunit in Human embryonic kidney (HEK)-293T cells and isolating complexes containing the Strep peptide-tagged BBS7 using a Strep-Tactin column. This purification resulted in roughly equal amounts of BBS2, BBS7, and BBS9 with a 70-kDa contaminant protein corresponding to heat shock protein 70 (Hsp70) isoforms (Fig. 1A)
Summary
Bardet-Biedl syndrome (BBS) is a genetic disorder characterized by malfunctions in primary cilia resulting from mutations that disrupt the function of the BBSome, an 8-subunit complex that plays an important role in protein transport in primary cilia. In this membrane-bound state, the BBSome is believed to pick up membrane proteins targeted for exit from the cilium [11, 18] Transport of these cargos occurs through association of the BBSome with the IFT-B complex via an interaction with a linker protein LZTFL1 (leucine zipper transcription factor-like 1) that binds to IFT27 in the IFT-B complex [15, 16, 20]. The two other subunits of the BBSome, BBS2 and BBS7, have an intricate folding and assembly process that requires a network of molecular chaperones, including the cytosolic chaperonin containing tailless polypeptide 1 (CCT; termed TRiC) complex and three chaperonin-like proteins named BBS6, -10, and -12 [22, 23]. A BBS-causing mutation in BBS2 (R632P) (29 –31) in this region disrupts the interaction of BBS2 with BBS9, suggesting that the inability of the BBS2/BBS7 dimer to associate with the hexameric complex is the underlying cause of BBS in patients with the R632P mutation
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