Structural analysis of human Cdc20 supports multisite degron recognition by APC/C

Abstract

The anaphase-promoting complex/cyclosome (APC/C) promotes anaphase onset and mitotic exit through ubiquitinating securin and cyclin B1. The mitotic APC/C activator, the cell division cycle 20 (Cdc20) protein, directly interacts with APC/C degrons––the destruction (D) and KEN boxes. APC/C Cdc20 is the target of the spindle checkpoint. Checkpoint inhibition of APC/C Cdc20 requires the binding of a BubR1 KEN box to Cdc20. How APC/C recognizes substrates is not understood. We report the crystal structures of human Cdc20 alone or bound to a BubR1 KEN box. Cdc20 has a disordered N-terminal region and a C-terminal WD40 β propeller with a preformed KEN-box-binding site at its top face. We identify a second conserved surface at the side of the Cdc20 β propeller as a D-box-binding site. The D box of securin, but not its KEN box, is critical for securin ubiquitination by APC/C Cdc20 . Although both motifs contribute to securin ubiquitination by APC/C Cdh1 , securin mutants lacking either motif are efficiently ubiquitinated. Furthermore, D-box peptides diminish the ubiquitination of KEN-box substrates by APC/C Cdh1 , suggesting possible competition between the two motifs. Our results indicate the lack of strong positive cooperativity between the two degrons of securin. We propose that low-cooperativity, multisite target recognition enables APC/C to robustly ubiquitinate diverse substrates and helps to drive cell cycle oscillations.

The Cdc20-binding Phe Box of the Spindle Checkpoint Protein BubR1 Maintains the Mitotic Checkpoint Complex During Mitosis

“…Protein Expression and Purification-The full-length Cdc20, Cdc20 WD40 (residues 161-477), and Mad2 L13A proteins were purified as previously described (16,32). The coding regions of various fragments of the human BubR1 middle region (BubR1M) were cloned into the pGEX-6p vector (GE Healthcare).…”

“…Structural studies of Cdc20 and Cdh1 have revealed similar APC/C degron-binding surfaces in these activators (15)(16)(17). They each contain a single, highly conserved KEN box-binding site on the top face of the ␤ propeller formed by their WD40 domain and a D box-binding site at the side of the ␤ propeller (Fig.…”

“…APC/C substrates are recognized through multiple, short degradation motifs called degrons, such as the destruction box (D box) and the KEN box (8). The KEN box is recognized by Cdc20 or its homolog Cdh1 (15)(16)(17), whereas the D box binds at the interface between Cdc20 (or Cdh1) and the core APC/C subunit APC10 (18,19). The MCC component BubR1 (or its yeast ortholog Mad3) contains two KEN boxes, which are required for checkpoint function and compete with KEN box-containing substrates for Cdc20 binding (20 -23).…”

“…Using a seat belt-like structural element, Mad2 in its active, closed conformation traps the Mad2-interacting motif of Cdc20 in a topological embrace (26 -28). The N-terminal region of BubR1 (BubR1N) contains two KEN boxes and interacts directly with Cdc20 through the first KEN box (KEN1) (16,23). Cdc20-bound Mad2 also makes direct contact with Mad3 (and quite possibly BubR1N) (17,29), buttressing the weak Cdc20-KEN1 interaction.…”

The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing

“…The protein p31 binds to Mad2 and removes it from the MCC leading to sub stoichiometric levels of Mad2 in the MCC 21 . An N-terminal KEN box motif in BubR1 is critical for stable binding to Cdc20 and binds to conserved residues on the top side of the WD40 domain 17,19,[22][23][24][25] . BubR1 in vertebrates also contains an internal Cdc20 binding domain (referred to here as IC20BD) that binds the Cdc20 WD40 domain in a Mad2-independent manner 6,26,27 .…”