APC/C inhibition
The mechanism by which MCC inhibits APC/C has not yet been defined at the atomic level. The KEN-box in Mad3 seems to act as a pseudo-substrate inhibitor of Cdc20 by blocking its degron-recognition site – this is when the inhibitor mimics the substrate and competes for the enzyme.[7] The KEN box interacts with the Cdc20 W40 domain through charged residues, through charge-charge interactions or and polar residues through polar contacts (Figure 4.1). When bound to co-activator, Cdc20, the KEN box has an underwound helical structure.
Figure 4.1 Polar interactions between the KEN box and Cdc20 W40 domain
Figure 4.2 Surface representation of Cdc20 with the binding configuration of the D-box mimic.
The D-box motif can be seen through the D-box mimic, the C-terminal of Mad 3 as an extended structure (Figure 4.2). The D-box motif alternately binds to the surface through its different amino acid side chains such as, the leucine residue (L215). The D-box binding channel is where degron recognition takes place and this is optimized once bound to negative residues, such as, arginine which is found in the arginine site.
Mutation of Cdc20’s residues responsible for KEN-box and D-box recognition and Mad3-binding results in no ubiquitination of substrates securin and Clb2. This is because the KEN and D-boxes are also present in APC/C’s substrates, securin and Clb2. Thus, understanding MCC's inhibition mechanism would lead to further insight into how Cdc20 activates APC/C, as the two processes are similar. Moreover, recent studies suggest MCC is able to inhibit a second Cdc20 already bound to APC/C in a mechanism that can occur without kinetochore signalling.[5]
Can the assembly be disrupted?
In short, yes - p31comet (found in animalia) competes for the same site on C-Mad 2 interface as Mad 3, interfering with its functionality within the MCC. After p31comet is bound to Mad2, it is able to inhibit the activation of the open state of Mad2 (O-Mad2). Thus a complex, C-Mad2-p31comet is made, stabilized through hydrophobic interactions (Figure 4.3). This promotes the disassembly of the MCC which then inhibits SAC activity. Consequently, this can lead to aneuploidy [3], an unusual number of chromosomes in the cell, which is closely associated with tumour formation.[2]
Figure 4.3 Interaction between p31comet and C-Mad2 through hydrophobic residues
I love the acronym list that reminds me the function of each component/feature in MCC. The introduction is also very well written as it gives us the biological context in which MCC operates in. However, it will be even better if you mention what p31 is and why does it induce aneuploidy in yeast. I am also quite curious why does the fission yeast protein is expressed in insect cell line.
ReplyDeleteThanks for your comment! I assume that the fission yeast protein is expressed in insect cell lines because post-translational modifications take place within the MCC that enables it to assemble and disassemble. Insect cell lines are known to be good at expressing large amounts of proteins with these post-translational modifications. Despite this, it is questionable as to why yeast cell lines such as Saccharomyces cerevisiae and Pichia pastoris weren't used instead.
DeleteFission yeast has actually been comparable to Metazoa (multicellular organisms) than budding yeast because of their protein similarity to mammalian proteins. Thus, there would be better expression of the fission yeast MCC in an insect cell line as opposed to yeast cell lines.
Also, p31comet is a protein that inhibits Mad2 because they are structurally similar. However, they differs slightly in structure as p31comet has helices that are connected by a disordered loop and short helix (possibly a π helix). Whereas, Mad 2's helices are connected via β8’ and β8” (seen from 'General Structure'). Because of these differences, it enables p31comet to inhibit Mad 2 effectively. When Mad 2 is inhibited, the MCC is inhibited. Therefore chromosomes could potentially be separated incorrectly which can lead to genomic instability like aneuploidy. Hope that helps! :)
Great webpage! There is a very good use of pymol, the diagrams are very clear, color coded and the relevant amino acid residues are properly highlighted. The text is very clear and well organized! The acronym list on the side is also very useful.
ReplyDelete