KEN and D box motifs

KEN and D-box motifs are degrons, specific sequences that control where degradation occurs. [6] The effect of degron binding sites on the inhibition on APC/C was analysed by using specific peptides that contained KEN-box and D-box motifs.

KEN-D peptides were generated, where peptides with KEN and D boxes were linked by 17 residues. This enables both KEN and D-boxes to cooperatively bind to their binding sites at a higher affinity - way higher than if they weren't attached to a peptide. In contrast, peptides with the reverse orientation of D and KEN boxes (D-KEN peptide) reduced the affinity of degron binding. Therefore, only one degron is capable of binding at the site in Cdc20.

Figure 3.  A comparison of securin levels using ubiquitination assays of D-KEN and KEN-D peptides. Figure taken from [4]

Figure 3 shows KEN-D peptide (lane 10) competitively inhibiting securin ubiquitation by APC/CCdh1 five times more than D-KEN peptide (lane 5). This is because KEN-D peptide can bind to the binding sites as both degrons are active. It can be inferred that cooperative degron binding occurs due to the specific spacial arrangement of the KEN and D boxes of KEN-D peptide because D-KEN peptide has the same reduced efficiency as D-box peptide in inhibiting APC/CCdh1. This spacial arrangement also agrees with the location of the binding sites on co-activators.