Molecular architecture of the Bardet¿Biedl syndrome protein 2-7-9 subcomplex

© 2019 Ludlam et al.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

Structural and functional analysis of the role of the chaperonin CCT in mTOR complex assembly

The mechanistic target of rapamycin (mTOR) kinase forms two multi-protein signaling complexes, mTORC1 and mTORC2, which are master regulators of cell growth, metabolism, survival and autophagy. Two of the subunits of these complexes are mLST8 and Raptor, β-propeller proteins that stabilize the mTOR kinase and recruit substrates, respectively. Here we report that the eukaryotic chaperonin CCT plays a key role in mTORC assembly and signaling by folding both mLST8 and Raptor. A high resolution (4.0 Å) cryo-EM structure of the human mLST8-CCT intermediate isolated directly from cells shows mLST8 in a near-native state bound to CCT deep within the folding chamber between the two CCT rings, and interacting mainly with the disordered N- and C-termini of specific CCT subunits of both rings. These findings describe a unique function of CCT in mTORC assembly and a distinct binding site in CCT for mLST8, far from those found for similar β-propeller proteins.This research was supported by the grant BFU2016-75984 (AEI/FEDER, EU) and the Madrid Regional Government (grant S2013/MIT2807) to J.M.V. as well as the US National Institutes of Health grant EY012287 to BMW and fellowships from the Brigham Young University Simmons Center for Cancer Research to W.G.L., N.C.T., T.A. and M.D

CBP/p300 are bimodal regulators of Wnt signaling

Many Wnts influence cell behavior by a conserved signaling cascade that promotes the stabilization and nuclear accumulation of β-catenin (β-cat), which then associates with TCF family members to activate target genes. The histone acetyltransferase CREB binding protein (CBP) can bind to TCF and inhibit Wnt signaling in Drosophila. In contrast, studies in vertebrates indicate a positive role for CBP and the closely related protein p300 as β-cat binding transcriptional co-activators. We address this discrepancy by demonstrating that in addition to its negative role, CBP has an essential positive role in Wnt signaling in flies. CBP binds directly to the C-terminus of Armadillo (Arm, the fly β-cat) and is recruited to a Wnt-regulated enhancer (WRE) in a Wnt- and Arm-dependent manner. In a human colorectal cancer cell line, we show that CBP and p300 can inhibit Wnt signaling and demonstrate that human p300 can bind directly to TCF4 in vitro. Our results argue that CBP/p300 has an evolutionarily conserved role as a buffer regulating TCF-β-cat/Arm binding. Subsequent to this interaction, it also has an essential role in mediating the transactivation activity of β-cat/Arm