Atomic structure of the APC/C and its mechanism of protein ubiquitination

Abstract

The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex, and interphase inhibitor Emi1 ensures the correct order and timing of distinct cell cycle transitions. Here, we used cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module, and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of experiments to further investigate APC/C functions in vivo.The activities of the diverse proteins that orchestrate the sequential biochemical and morphological changes intrinsic to the cell division cycle are controlled through the integration of protein phosphorylation, proteolysis and changes in gene expression. Two cullin-RING E3 ubiquitin ligases, the APC/C and SCF, catalyze the ubiquitination of multiple cell cycle proteins to regulate their proteasome-mediated proteolysis. By regulating the ordered degradation of substrates such as cyclins, securin, mitotic kinases, and microtubule motors and assembly factors, the APC/C controls sister chromatid segregation, cytokinesis and the initiation of chromosome duplication [1][2][3] .The APC/C is a large assembly comprising 19 subunits 4 . Its activity depends on the association of one of two coactivator subunits (either Cdc20 or Cdh1) that specify substrate recognition and stimulate the catalytic activity of APC/C -E2 complexes [4][5][6][7] . Coactivators engage the APC/C through a conserved N-terminal C box motif and a C-terminal Ile-Arg Correspondence should be addressed to D.B. (dbarford@mrc-lmb.cam.ac.uk). Author contributions. L.C. prepared grids, collected and analyzed EM data and determined the 3D reconstructions, fitted coordinates and built models, prepared figures, co-wrote the paper. Z.Z. designed and made constructs, performed biochemical analysis and purified proteins. J.Y. prepared and purified the complexes and performed biochemical analysis. S.H.McL. performed and analyzed SPR experiments. D.B. directed the project, built models and co-wrote the paper.Author information. EM maps are deposited with the EM-DB with accession codes: 2924 (APC/C Cdh1.Emi1 ), 2925 (APC/ C Cdh1.Hsl1.UbcH10-Ub) , 2926 (APC/C Cdh1.Hsl1.Apc11-UbcH10) . APC/C Cdh1.Emi1 coords have accession code 4ui9.The authors declare no competing financial interests. Europe PMC Funders GroupAuthor Manuscript Nature. Author manuscript; available in PMC 2015 December 25. Published in final edited form as:Nature. 2,3 . Cyclin-dependent kinase (CDK) phosphorylates the...

WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity

“…This change in conformation exposes the UbcH10-binding site of the catalytic module, enhancing UbcH10 association and thereby stimulating APC/C E3 ligase activity (12,19). Here, we combined biochemical methods, X-ray crystallography, and single-particle EM to study the structure of Apc1N and to examine its functions in vitro.…”

“…1A. Apc1N is followed by the middle domain (Mid-N), a PC domain, and a C-terminal domain (Mid-C) that coalesces with Mid-N to form Apc1 Mid (19). Combining the secondary structure predictions of Apc1 with structural information from the APC/C atomic model determined using a 3.6-Å resolution cryo-EM map (19), we designed the following Apc1N constructs: Apc1N WT , Apc1N Δloop2 , Apc1N Δloops2&3 , and Apc1N Δloops1&2&3 (Fig.…”

“…The crystal structure of Apc1N Δloops1&2&3 was determined using the cryo-EM-derived APC/C atomic model (19) as a search object in molecular replacement. The crystal structure of Apc1N Δloops1&2&3 was confirmed as a WD40 β-propeller domain, which consists of seven blades, each with either four or five β-strands (Fig.…”

“…The association of UbcH10 with the RING domain of Apc11 (Apc11 RING ) and Apc2's winged-helix B domain (Apc2 WHB ) initiates ubiquitin-chain formation (19,20,(24)(25)(26). Ube2S, on the other hand, is responsible for ubiquitin-chain extension and specifically assembles Lys11-linked ubiquitin chains on substrates targeted by the APC/C (14,25,(27)(28)(29)(30).…”

“…The N-terminal region of Apc1 (Apc1N) is rich in β-strands and possesses a multitude of regulatory phosphorylation sites (17,18). The APC/C catalytic module composed of Apc2 and Apc11 recruits canonical E2s (UbcH10 and UbcH5 in vertebrates, Ubc1 and Ubc4 in budding yeast) to catalyze substrate ubiquitination (19,20). Apc2 is a cullin domain protein that interacts with the RING domain subunit Apc11.…”