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Structural Analysis of Human Cdc20 Supports Multisite Degron Recognition by APC/C

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Structural Analysis of Human Cdc20 Supports Multisite Degron Recognition by APC/C Structural analysis of human Cdc20 supports multisite degron recognition by APC/C Wei Tiana,1, Bing Lia,b,1, Ross Warringtona,b, Diana R. Tomchickc, Hongtao Yua,b,2, and Xuelian Luoa,2 aDepartment of Pharmacology, bHoward Hughes Medical Institute, and cDepartment of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390 Edited by Stephen C. Harrison, Howard Hughes Medical Institute and Children’s Hospital, Harvard Medical School, Boston, MA, and approved September 24, 2012 (received for review August 3, 2012) The anaphase-promoting complex/cyclosome (APC/C) promotes suggested to inhibit APC/C by acting as a pseudosubstrate, with anaphase onset and mitotic exit through ubiquitinating securin its KEN boxes competing against those of the substrates for and cyclin B1. The mitotic APC/C activator, the cell division cycle 20 Cdc20 binding (22, 23). (Cdc20) protein, directly interacts with APC/C degrons––the de- To better understand how APC/C recognizes its substrates, struction (D) and KEN boxes. APC/CCdc20 is the target of the spindle we determined the crystal structures of human Cdc20 alone or checkpoint. Checkpoint inhibition of APC/CCdc20 requires the bind- bound to the KEN1 box of BubR1, revealing a preexisting KEN- box-binding site at the top of the WD40 β propeller. Structure- ing of a BubR1 KEN box to Cdc20. How APC/C recognizes sub- based mutagenesis then defined a second conserved surface on strates is not understood. We report the crystal structures of the side of the β propeller as a D-box-contacting site. Surpris- human Cdc20 alone or bound to a BubR1 KEN box. Cdc20 has ingly, despite binding to distinct sites on Cdc20, the KEN and D a disordered N-terminal region and a C-terminal WD40 β propeller boxes of securin do not apparently interact with APC/C in a with a preformed KEN-box-binding site at its top face. We identify highly cooperative manner. The KEN box is largely dispensable a second conserved surface at the side of the Cdc20 β propeller as for securin ubiquitination by APC/CCdc20. This lack of strong a D-box-binding site. The D box of securin, but not its KEN box, is cooperativity between the two degrons also applies to APC/ critical for securin ubiquitination by APC/CCdc20. Although both CCdh1, but the KEN and D boxes have comparable contributions motifs contribute to securin ubiquitination by APC/CCdh1, securin in this case. D-box peptides inhibit APC/C-mediated ubiquiti- mutants lacking either motif are efficiently ubiquitinated. Further- nation of KEN-box substrates, suggesting competition between BIOPHYSICS AND the two motifs. Our results indicate that the multiple APC/C more, D-box peptides diminish the ubiquitination of KEN-box sub- COMPUTATIONAL BIOLOGY strates by APC/CCdh1, suggesting possible competition between degrons of securin do not engage APC/C in a highly cooperative manner. A single functional APC/C degron within a substrate the two motifs. Our results indicate the lack of strong positive Cdh1 cooperativity between the two degrons of securin. We propose supports APC/C -dependent ubiquitination. We propose that that low-cooperativity, multisite target recognition enables APC/C this low-cooperativity, multisite substrate recognition mechanism expands the substrate spectrum of APC/C, allowing it to atten- to robustly ubiquitinate diverse substrates and helps to drive cell uate the cellular concentrations of myriad substrates with one or cycle oscillations. more functional degrons. mitosis | crystallography | multivalency | molecular recognition Results and Discussion Structures of Human Cdc20 by Itself or in Complex with BubR1 KEN1. he anaphase-promoting complex/cyclosome (APC/C) is a To determine the structure of human Cdc20, we expressed and Tmultisubunit ubiquitin ligase that mediates the ubiquitination purified from Sf9 insect cells a Cdc20 truncation mutant with its Δ Δ of a multitude of substrates to drive cell cycle progression (1–3). N-terminal 60 residues deleted (Cdc20 N60). Cdc20 N60 con- At the metaphase–anaphase transition, APC/C in complex with tained all known functional motifs of Cdc20 (3), including the C its mitotic activator Cdc20 (APC/CCdc20) ubiquitinates securin box, the KEN box, and the Mad2-interacting motif (MIM), and and cyclin B1, triggering their destruction to allow separase ac- actively supported APC/C- and D-box-dependent ubiquitination tivation and cohesin cleavage. In anaphase, APC/C bound to the of cyclin B1 (Fig. 1A and Fig. S1). We obtained crystals of Cdh1 Δ Cdc20 homolog Cdh1 (APC/C ) further mediate the ubiq- Cdc20 N60 that diffracted to about 3 Å, but could not determine uitination of cyclin B1 and other mitotic regulators, resulting in its structure using molecular replacement. We thus made addi- Δ Δ their degradation and mitotic exit. tional Cdc20 truncation mutants Cdc20 N70 and Cdc20 N80, Δ Cdc20 and Cdh1 activate APC/C by contributing to substrate obtained crystals of Cdc20 N80 that diffracted to 2.0 Å, and recognition, among other mechanisms (1–3). All APC/C substrates determined its structure using molecular replacement with the contain one or more of several types of short peptide motifs with coordinates of Wdr5 as the search model (Table S1). Only the Δ highly degenerate sequences, referred to as APC/C degrons. For WD40 domain of Cdc20 N80 was well defined in the structure. example, cyclin B1 contains a destruction (D) box with the con- Both the N-terminal region (residues 81–164) and the C-terminal ΔN80 sensus of RXXLX4–5N (X, any residue), whereas securin contains region (residues 477–499; including the IR motif) of Cdc20 both a D box and a KEN box (4-6). Biochemical and biophysical were disordered and not visible in the structure. We then obtained Δ studies have established that Cdh1 and the APC/C subunit Apc10 an unrefined structure model of Cdc20 N60 using molecular serve as coreceptors for the D box, with the D box bridging an interaction between the two (7–11). APC/CCdc20 is the molecular target of the spindle checkpoint, which prevents premature sister-chromatid separation and an- Author contributions: W.T., B.L., R.W., and X.L. performed research; D.R.T., H.Y., and X.L. – analyzed data; H.Y. and X.L. designed research; and H.Y. and X.L. wrote the paper. euploidy (3, 12 15). In response to kinetochores not properly fl attached to the spindle microtubules, the spindle checkpoint The authors declare no con ict of interest. inhibits APC/CCdc20 using multiple mechanisms. Among these This article is a PNAS Direct Submission. mechanisms, the mitotic checkpoint complex (MCC; consisting Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, of BubR1/Mad3, Bub3, Mad2, and Cdc20) anchors Cdc20 at a www.pdb.org (PDB ID codes 4GGA, 4GGC, and 4GGD). site on APC/C that is different from the site bound by free 1W.T. and B.L. contributed equally to this work. Cdc20, presumably blocking the productive engagement of the D 2To whom correspondence may be addressed. E-mail: [email protected] or box with Cdc20 and Apc10 (16-21). BubR1/Mad3 contains two [email protected]. KEN boxes (KEN1 and KEN2), both of which are required for This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the spindle checkpoint (22–25). BubR1/Mad3 has thus been 1073/pnas.1213438109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1213438109 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Fig. 1. Human Cdc20 has a preformed KEN-box- binding site at the top face of its WD40 β propeller. (A) Domains and motifs of human Cdc20. MIM, Mad2 Interacting Motif. (B) Ribbon diagram of hu- man Cdc20. The N and C termini and the seven blades of the WD40 β propeller are labeled. The DYY insert in the loop connecting blades 1 and 7 is colored salmon. All structure figures in this report are generated with PyMOL (www.pymol.org). (C) Ribbon diagram of Wdr5 (PDB ID code 2GNQ). (D) Surface drawing of the Cdc20ΔN70–BubR1 KEN1 complex with BubR1 KEN1 shown as sticks. The DYY insert is colored salmon. (E) Surface drawing of the Wdr5–histone H3 complex (PDB ID code 2H9O), with the bound H3 peptide shown as sticks. (F) A cross- sectional view of the surface drawing of Cdc20– BubR1 KEN1 in D.(G) A cross-sectional view of the surface drawing of Wdr5–H3 in E. Δ replacement with Cdc20 N80 as the search model, but could not of human BubR1. The structures of free and KEN-bound Cdc20 refine the structure due to poor quality diffraction data. were virtually identical, indicating that KEN-box binding did not Nonetheless, it was clear that the N- and C-terminal regions of induce conformational changes of Cdc20. Only eight residues of Δ the functional Cdc20 N60 (residues 61–164 and 477–499) were the KEN1 peptide (from E23 to Q30), including the KEN motif, also disordered. were visible in the structure (Fig. 1D). The KEN motif (residues The WD40 domain of Cdc20 expectedly folds into a canonical 26–28) folds into an irregular 310 helix that binds at the top face seven-bladed β propeller. Unlike Wdr5 and other WD40 domain of the propeller. A highly conserved DYY motif of the afore- structures (26), Cdc20 does not have a deep ligand-binding mentioned insert of Cdc20 establishes the base of the KEN-box- pocket at the top face of its β propeller (Fig. 1 B–G). Instead, an binding site, contacting the KEN backbone and the side chains of insert (containing residues 183–188) in the loop connecting the E27 and N28 (Figs.
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