Cronin NB et al. (2015), Atomic-Resolution Structures of the APC/C Subun...

XB-ART-53314

J Mol Biol 2015 Oct 09;42720:3300-3315. doi: 10.1016/j.jmb.2015.08.023.

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Atomic-Resolution Structures of the APC/C Subunits Apc4 and the Apc5 N-Terminal Domain.

Cronin NB , Yang J , Zhang Z , Kulkarni K , Chang L , Yamano H , Barford D .

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Many essential biological processes are mediated by complex molecular machines comprising multiple subunits. Knowledge on the architecture of individual subunits and their positions within the overall multimeric complex is key to understanding the molecular mechanisms of macromolecular assemblies. The anaphase-promoting complex/cyclosome (APC/C) is a large multisubunit complex that regulates cell cycle progression by ubiquitinating cell cycle proteins for proteolysis by the proteasome. The holo-complex is composed of 15 different proteins that assemble to generate a complex of 20 subunits. Here, we describe the crystal structures of Apc4 and the N-terminal domain of Apc5 (Apc5(N)). Apc4 comprises a WD40 domain split by a long α-helical domain, whereas Apc5(N) has an α-helical fold. In a separate study, we had fitted these atomic models to a 3.6-Å-resolution cryo-electron microscopy map of the APC/C. We describe how, in the context of the APC/C, regions of Apc4 disordered in the crystal assume order through contacts to Apc5, whereas Apc5(N) shows small conformational changes relative to its crystal structure. We discuss the complementary approaches of high-resolution electron microscopy and protein crystallography to the structure determination of subunits of multimeric complexes.

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Species referenced: Xenopus laevis
Genes referenced: anapc1 apc2 fbxo5 tpr

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	Graphical Abstract
	Fig. 1. Apc4 comprises a WD40 β-propeller toroid split by a helical bundle domain. (a) Cartoon of Apc4 color-ramped from blue to red from N- to C-termini. Shown is the EM structure of human Apc4. (b) Close-up view of the extended blade 5 of Apc4WD40 and showing the βD4/βA5 loop that blocks access to the mouth of the WD40 domain tunnel. (c) Stereoview showing that the M-domain of α-catenin, superimposed onto the EM structure of human Apc4HBD, shares structural similarity with the four-helical-bundle domain of Apc4.
	Fig. 2. The Apc4–Apc5 protein interface orders regions of Apc4HBD. (a) Human Apc4–Apc5 as organized in the APC/C with the X-ray structure of Apc4 (red) superimposed onto the EM coordinates (brown). The Apc4–Apc5 interface is shown. (b) As in (a) but without Apc5. (c) Stereoview showing that the crystal structures of X. laevis and human Apc4 are very similar (RMSD is 2.1 Å).
	Fig. 3. Comparison of EM density maps of APC/CCdh1.Emi1 and crystal structure 2Fo − Fc maps of human Apc4. (a) Stereoview of the EM density map and Apc4 coordinates. Main chain is shown as a cartoon. (b) Stereoview of the 2Fo − Fc density map contoured at 1σ and Apc4 coordinates. Main chain is shown as a cartoon.
	Fig. 4. Comparison of EM density maps of APC/CCdh1.Emi1 and crystal structure 2Fo − Fc maps of human Apc4HBD. (a) Stereoview of the EM density map and Apc4 coordinates. Main chain is shown as a cartoon, and amino acid side chains are shown as sticks. (b) Stereoview of the 2Fo − Fc density map contoured at 1σ and Apc4 coordinates. Main chain is shown as a cartoon, and amino acid side chains are shown as sticks.
	Fig. 5. Comparison of EM density maps of APC/CCdh1.Emi1 and crystal structure 2Fo − Fc maps of human Apc4WD40. (a) Stereoview of the 2Fo − Fc density map contoured at 1σ and Apc4 coordinates. Main chain is shown as a cartoon, and amino acid side chains are shown as sticks. (b) Stereoview of the EM density map and Apc4 coordinates. Main chain is shown as a cartoon, and amino acid side chains are shown as sticks.
	Fig. 6. Apc5 has an N-terminal helical domain (Apc5N) connected by a disordered linker to a TPR superhelix of 13 TPR motifs (Apc5TPR). (a) Cartoon of EM structure of human Apc5 color-ramped from blue to red from N- to C-termini. The small subunit Apc15 that contacts Apc5 is also shown. (b) Stereoview of a superimposition of Apc5N based on human Apc5N EM coordinates (blue) and Xenopus Apc5N X-ray (yellow) coordinates. Major structural differences involve the α1/α2 and α5/α6 loops that contact Apc4.
	Fig. 7. Apc4 and Apc5 interact with neighboring subunits through evolutionarily conserved protein–protein interfaces. (a) Surface of Apc4 color-coded according to conservation (purple, conserved; cyan, unconserved). Contacting subunits Apc2, Apc5 and Apc15 are shown as cartoons. (b and c) Surface of Apc5 color-coded according to conservation (purple, conserved; cyan, unconserved). Contacting subunits Apc1, Apc4, Apc8 and Apc15 are shown as cartoons. (d) Cryo-EM density map of the APC/CCdh1.Hsl1–UbcH10 complex, color-coded according to subunit assignments [44]. Density connects Apc2WHB with the blade 3 insert of Apc4WD40, suggesting a direct interaction between these domains. EM coordinates are shown.

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