How co-activators recognize substrates and inhibitors

• In order to study co-activators, the research team designed peptides that contained KEN-box and D-box motifs. They then tested their ability to inhibit APC/C^cdh1 so they could assign the APC/C degron binding sites on co-activators further.

• A peptide which contains KEN and D boxes (KEN-D orientation) linked by 17 residues enables cooperative binding of both degrons to their respective binding sites. It has higher binding affinity than peptides with only 1 degron.

• A peptide with the reverse orientation (D-KEN) only allows one degron to bind.

• A KEN-D orientation in the peptide makes it 5 times more effective as an inhibitor than D-KEN when competitively inhibiting securin ubiquination by APC/C^cdh1.

• Co-operative degron binding to cdh1 is determined by the spatial arrangement of the KEN and D boxes of the peptide; as the D-KEN peptide inhibits APC/C^cdh1 with the same efficiency as a D-box peptide.

• KEN and D box domains arrange in different conformations when bound to cdc20, Ken forms an underwound helix whilst D box takes on an extended structure [3].

• This means that the 3 residues of the KEN domain are presented to the same surface on cdc20, whilst the D box alterntive amino acid side chains are oriented in opposite directions.    

•  Therefore as the D box is anchored by the co-activator via the D box’s Leucine side chain; Arg, Ile/Leu and Asn side chains of the D box are available to form a composite D box co-activator recognition surface for Apc10 (D-box co receptor) [4,5].

• The spatial arrangement of KEN and D boxes in the KEN-D inhibitory peptide is similar to that of 2 APC/C inhibitors: Acm1 and Mes1. They can both inhibit APC/C co-activators thanks to their KEN and D boxes [6-8].