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

Area of Science:

• Cell Biology
• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• The anaphase-promoting complex/cyclosome (APC/C) is a crucial E3 ubiquitin ligase complex regulating cell cycle progression.
• APC/C activity is tightly controlled by activators like Cdc20 and Cdh1, and targets substrates like securin and cyclin B1 for degradation.
• Substrate recognition by APC/C involves degrons, primarily destruction (D) boxes and KEN boxes, but the precise mechanisms remain unclear.

Purpose of the Study:

• To elucidate the structural basis of APC/C substrate recognition by Cdc20.
• To understand how APC/C distinguishes between different degron motifs (D-box and KEN box) on its substrates.
• To investigate the role of these motifs in substrate ubiquitination and APC/C regulation.

Main Methods:

• Crystal structures of human Cdc20, both alone and bound to a BubR1 KEN box peptide.
• Biochemical assays to assess the ubiquitination of securin mutants lacking D-box or KEN-box motifs.
• Peptide competition assays to study the interaction between D-box and KEN-box motifs.

Main Results:

• Crystal structures revealed Cdc20 possesses a KEN-box binding site on its WD40 propeller and identified a separate D-box binding site.
• The D-box of securin, but not its KEN box, is essential for ubiquitination by APC/C(Cdc20).
• D-box peptides can inhibit the ubiquitination of KEN-box substrates by APC/C(Cdh1), suggesting motif competition.

Conclusions:

• APC/C utilizes distinct, potentially competitive binding sites for D-boxes and KEN boxes, enabling flexible substrate recognition.
• Low cooperativity between degrons allows APC/C to efficiently ubiquitinate diverse substrates, contributing to robust cell cycle oscillations.
• Understanding these recognition mechanisms provides insight into cell cycle control and potential therapeutic targets.