Sandeep K Singh, Miao Gui, Fujiet Koh, Matthew CJ Yip, and Alan Brown. 2020. “Structure and activation mechanism of the BBSome membrane protein trafficking complex.” Edited by Andrew P Carter. eLife, 9, Pp. e53322. Publisher's VersionAbstract
Bardet-Biedl syndrome (BBS) is a currently incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS assemble into the BBSome, a key regulator of the ciliary membrane proteome. We report the electron cryomicroscopy (cryo-EM) structures of the native bovine BBSome in inactive and active states at 3.1 ­and 3.5 Å resolution, respectively. In the active state, the BBSome is bound to an Arf-family GTPase (ARL6/BBS3) that recruits the BBSome to ciliary membranes. ARL6 recognizes a composite binding site formed by BBS1 and BBS7 that is occluded in the inactive state. Activation requires an unexpected swiveling of the b-propeller domain of BBS1, the subunit most frequently implicated in substrate recognition, which widens a central cavity of the BBSome. Structural mapping of disease-causing mutations suggests that pathogenesis results from folding defects and the disruption of autoinhibition and activation.
Eun Young Park, Shaun Rawson, Kunhua Li, Byeong-Won Kim, Scott B. Ficarro, Gonzalo Gonzalez-Del Pino, Humayun Sharif, Jarrod A. Marto, Hyesung Jeon, and Michael J. Eck. 2019. “Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes.” Nature. Publisher's VersionAbstract
RAF family kinases are RAS-activated switches that initiate signaling through the MAP kinase cascade to control cellular proliferation, differentiation and survival1-3. RAF activity is tightly regulated, and inappropriate activation is a frequent cause of cancer4-6. At present, the structural basis of RAF regulation is poorly understood. Here we describe autoinhibited and active state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer, determined using cryo-electron microscopy (cryo-EM). A 4.1?\AA resolution cryo-EM reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain (CRD) occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain (RBD) is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding CRD and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, driving formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.
Stephen M Hinshaw, Andrew N Dates, and Stephen C Harrison. 2019. “The structure of the yeast Ctf3 complex.” Edited by Andrea Musacchio. eLife, 8, Pp. e48215. Publisher's VersionAbstract
Kinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture (Hinshaw and Harrison, 2019). We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.
Edward C. Twomey, Zhejian Ji, Thomas E. Wales, Nicholas O. Bodnar, Scott B. Ficarro, Jarrod A. Marto, John R. Engen, and Tom A. Rapoport. 2019. “Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding.” Science. Publisher's VersionAbstract
The Cdc48 ATPase (p97 or VCP in mammals) and its cofactor Ufd1/Npl4 extract poly-ubiquitinated proteins from membranes or macromolecular complexes for subsequent degradation by the proteasome. How Cdc48 processes its diverse and often well-folded substrates is unclear. Here, we report cryo-EM structures of the Cdc48 ATPase in complex with Ufd1/Npl4 and poly-ubiquitinated substrate. The structures show that the Cdc48 complex initiates substrate processing by unfolding a ubiquitin molecule. The unfolded ubiquitin molecule binds to Npl4 and projects its N-terminal segment through both hexameric ATPase rings. Pore loops of the second ring form a staircase that acts as a conveyer belt to move the polypeptide through the central pore. Inducing the unfolding of ubiquitin allows the Cdc48 ATPase complex to process a broad range of substrates.
Chao Fan, Minrui Fan, Benjamin J. Orlando, Nathan M. Fastman, Jinru Zhang, Yan Xu, Melissa G. Chambers, Xiaofang Xu, Kay Perry, Maofu Liao, and Liang Feng. 7/11/2018. “X-ray and cryo-EM structures of the mitochondrial calcium uniporter.” Nature, 559, Pp. 575-579.
Yibei Xiao, Min Luo, Adam E. Dolan, Maofu Liao, and Ailong Ke. 7/6/2018. “Structure basis for RNA-guided DNA degradation by Cascade and Cas3.” Science, 361, 6397.
Anura P. Srivastava, Min Luo, Wenchang Zhou, Jindrich Symersky, Dongyang Bai, Melissa G. Chambers, Jose D. Faraldo-Gomez, Maofu Liao, and David M. Mueller. 5/11/2018. “High-resolution cryo-EM analysis of the yeast ATP synthase in a lipid membrane.” Science, 360, 6389.
Wei Mi, Yanyan Li, Sung Hwan Yoon, Robert K. Ernst, Thomas Walz, and Maofu Liao. 9/14/2017. “Structural basis of MsbA-mediated lipopolysaccharide transport.” Nature, 549, Pp. 233-237.
Bart Alewijnse, Alun W. Ashton, Melissa G. Chambers, Songye Chen, Anchi Cheng, Mark Ebrahim, Edward T. Eng, Win J.H. Hagen, Abraham J. Koster, Claudia S. Lopez, Natalya Lukoyanova, Joaquin Ortega, Ludovic Renault, Steve Reyntjens, William J. Rice, Giovanna Scapin, Raymond Schrijver, Alistair Siebert, Scott M. Stagg, Valerie Grum-Tokars, Elizabeth R. Wright, Shenping Wu, Zhiheng Yu, Hong Zhou, Bridget Carragher, and Clinton S. Potter. 9/2017. “Best practices for managing large CryoEM facilities.” Journal of Structural Biology, 199, 3, Pp. 225-236.
Yibei Xiao, Min Luo, Robert B. Hayes, Jonathan Kim, Sherwin Ng, Fang Ding, Maofu Liao, and Ailong Ke. 6/29/2017. “Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.” Cell, 170, 1, Pp. 48-60.
Heng Ru, Melissa G. Chambers, Tian-Min Fu, Alexander B. Tong, Maofu Liao, and Hao Wu. 11/5/2015. “Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures.” Cell, 163, 5, Pp. 1138-1152.