Structuur-functie analyse van een nieuwe plant specifieke inhibitor van het anafase-bevorderend complex

Cell division in higher organisms (Eukaryotes) is a tightly controlled process. A cell can divide only after completing two key processes: replicating its entire genome and segregating its double set of chromosomes. Cell division is halted at several specific stages, called checkpoints, and is only allowed to proceed if all necessary conditions are met. In this way, it is ensured that all daughter cells are healthy and their DNA is not damaged. Indeed, cells are programmed to die rather than creating abnormal cells. In one such checkpoint the anaphase promoting complex (APC/C) triggers the transition from metaphase to anaphase by tagging specific proteins with ubiquitin for degradation.
APC/C is a large multi-subunit complex who's activity is regulated though activators and inhibitors. For example, the activators Cdc20 and Cdh1 are required for substrate recognition through the so-called D and KEN boxes (short recognition sequences on APC/C substrates). Alternatively, the inhibitor Emi1 blocks Cdc20's interaction with APC targets via acting as a pseudo-substrate.
UVI4 is the archetype of a recently discovered family of APC/C inhibitors in plants, which have no clear homologs in animals. UVI4 nevertheless contains a D- and a GxEN-box, indicating that it also might act as a pseudo-substrate. In this PhD project I propose to study the structure of UVI4 through a combination of X-ray crystallography, SAXS and NMR spectroscopy. In addition, I will study the interaction between UVI4 and its co-activator CCS52A1 (the Arabidopsis homolog of Cdh1), APC4/5 and the complete APC/C complex. This will include ITC and/or SPR experiments to determine binding constants and other thermodynamic parameters as well as crystal structure determination of CCS52A1 and APC4/5 (the latter being the "platform" structure for which currently no molecular model is available), This will ultimately lead to a detailed understanding of substrate and inhibitor-recognition by APC/C, something for which structural an molecular details are lacking.