As seen in figure 2.1, the MCC crystal structure is a triangular
heterotrimer. This multi-subunit assembles in two steps. First, Mad2 and Cdc20 form
a dimer, then, Mad3 binds, stabilising the overall assembly by
forming numerous interactions with both subunits.
Figure 2.1 Assembly of crystal MCC structure using Pymol.
Cdc20
Cdc20 comprises 4 functional domains: WD40 domain, Mad2-binding motif, C-box and Ile-Arg tail. Cdc20, along with Cdh1, are APC/C co-activators, and by incorporating Cdc20 into the complex, MCC prevents APC/C activation, delaying anaphase.
The WD40 domain folds into a propeller of β-sheets with seven blades (repetitive regions). Within it are two well-conserved regions, both degron (destruction motif) receptors, able to recognise the D (destruction) box (this is shown as a D-box mimic) and KEN box on the APC/C.[7] However, these recognition sites are occupied by Mad3 in MCC.
The Mad2-binding motif (MB motif) reaches into the safety belt in Mad2 and is linked to the WD40 N-terminus via a highly disordered loop, which is why it is absent from the crystal structure. The MB motif interacts with the safety belt through electrostatic interactions, specifically between the nitrogens in the arginine residue (R128) and oxygens in aspartic acid (D169). Two additional disordered motifs interact with APC/C, respectively the C-terminal tail and a conserved C-box.
The Mad2-binding motif (MB motif) reaches into the safety belt in Mad2 and is linked to the WD40 N-terminus via a highly disordered loop, which is why it is absent from the crystal structure. The MB motif interacts with the safety belt through electrostatic interactions, specifically between the nitrogens in the arginine residue (R128) and oxygens in aspartic acid (D169). Two additional disordered motifs interact with APC/C, respectively the C-terminal tail and a conserved C-box.
Figure 2.2 Interaction of Cdc20's Mad2-binding motif with the Mad2 safety belt.
Mad2
Mad2 can exist in either open (O-Mad2) or closed (C-Mad2) conformation (Figure 2.3). C-Mad2 is the only conformation able to interact with Cdc20 and Mad3. The C-α-helix (residues 122–142) and β8’- β8’’hairpin interact with the Mad3 HLH motif.[8] Mutagenesis in C-α-helix dissociates Mad3 from the C-Mad2-Cdc20 dimer, showing it is essential for Mad3 binding to Cdc20 with high affinity. The β8’- β8’’ region significantly changes conformation between C-Mad2 and O-Mad2 states, forming the Mad2-Mad 3 interface.
Figure 2.3 Open (O-Mad2) and closed (C-Mad2) conformations of Mad2
PDB accession code: 2V64
Mad3
Mad3 is a tetratricopeptide-repeat (TPR) superhelix composed of 3 adjacent TPR motifs flanked by alpha helices. The TPR domain mediates protein-protein interactions with Cdc20 and Mad2 and thereby allows complex formation.A KEN-box (signalling motif with K, E, N residues) is incorporated into a conserved N-terminal helix-loop-helix (HLH) domain. Mad 3 HLH binds to Mad2 and Cdc20 and orientates the KEN box towards its Cdc20 receptor. Thus, E21 is located at the centre of an underwound helix, so it dips into the centre of the propeller on Cdc20. A hydrophobic interaction between E21's aliphatic side chain and Y181 from Cdc20 stabilises this conformation. Whereas, the rest of the KEN box forms charge-charge interactions with 4 loops from the WD40 propeller (Figure 2.4).
Figure 2.4 Mad3 HLH interaction with Mad2 and Cdc20 in cartoon configuration.
The authors have effectively used PyMOL in all its capacities, including effective colour coding and using transparency settings to effectively highlight the protein's most important functions and structure. I also like the way the PyMOL images are labelled which makes the process of understanding the protein structure much easier and effective.
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