WD40 Domain

KEN and D box Receptors:

·    Two highly conserved sequences responsible for the recognition of degrons on APC/C.

·    KEN box receptor on the top side of the WD40 domain, at the centre of the β-propeller

·    D box co-receptor found on the WD40 domain in a channel between blades 1 and 7 of the β-propeller.
·    Figure 2 shows residues K20-E21-N22, the KEN box of Mad3 - emerge from the C- terminus of α-A helix of the HLH motif on Mad3.

·    These residues take on a conformation which has a ‘U’-like shape with the middle residue, E21, at the turn of this ‘U’-like conformation dipping into a depression at the Cdc20’s β-propeller toroid centre

·    Y181 of Cdc20 located on an extended loop structure connecting blades 1 and 7 of the β-propeller acts as a platform for the aliphatic group of E21 – only hydrophobic interaction between KEN box and receptor.

·    Other contacts are entirely polar between side chains of KEN box residues and peptide backbone with the conserved polar and charged residues of the loops which are on four of the seven blades of the β-propeller.

·    D180, N326, T368, Q392 and R438 are all interacting residues on Cdc20 with the KEN box -invariant between Cdc20 and Cdh1 - indicates conserved mode of interaction, and Figure 2 indicates the contacts and their distances (in angstroms) involved.
·    P25 located 3 amino acids C-terminal to KEN box is conserved - function is break the α-helix by introducing a turn orientating the polypeptide chain so that it no longer faces the Cdc20 surface [2].



Figure 2, indicating the interaction of the KEN box (pink) with its receptor (orange) on the WD40 domain of Cdc20.



• It has been suggested that the Mad3 KEN box is a pseudosubstrate and acts as an inhibitor of APC/C by blocking the access of the Cdc20 WD40 to the KEN box degrons which are found on the substrates of APC/C - confirmed by mutagenesis studies.

The WD40 domain:

·         Interacts with the D box degron and thus acts as a co-receptor with Apc10

·         Unpredicted crystal packing contact found - Mad3 subunit’s carboxyl-terminus mimics a D box and contacts Cdc20, which provides some detailed molecular insights with regard to the recognition of the D box by co-activators.

·         D box mimic located in an extended conformation situated within a evolutionarily conserved channel located between blades one and seven of the β-propeller.

·         Interaction driven by the burial of Leucine 215 of Mad3 which is aliphatic within a deep pocket which complements the Leucine side chain and is created by non-polar amino acid residues which are found conserved in Cdc20 and Cdh1.

·         This mimics the Leucine of the D box RXXL motif. However, the other consensus residues of the D box motif which are the invariant Arginine at position 438 and the less conserved (I/L)(S/T)N motif are not found represented in the mimic of the D box in Mad3.

·         Despite this a potential recognition site for the Arginine 438 side chain of the D box can be allocated to a specific cluster of negatively charged residues, which are D173, D457 and E458 (highlighted pink in Figure 3), and these are located on two β-strands which are next to each other on blade seven of the β-propeller, on the top side of the D box motif binding channel.

·         The position of these residues is such that they are appropriately to interact with the postive Arginine residue, which is amino-terminal to the anchored Leucine 215 residue, and these residues are highly conserved in Cdh1 [2].

Figure 4. showing the negatively charged residues of Cdc20 which recognise the Arg side chain of the D box.