The chromosomal passenger complex (CPC: Aurora B-INCENP-Survivin-Borealin) is a key regulator of cell division whose localisation at centromeres is required for stable kinetochore-microtubule attachments and proper chromosomal segregation (Ruchaud et al. 2007; Carmena et al. 2012; van der Waal et al. 2012). Shugoshin1 (hSgo1) (via Borealin) and Histone H3 (via Survivin) have been implicated in centromeric targeting of CPC (Wang et al. 2010; Jeyaprakash et al. 2011; Tsukahara et al. 2010; Kawashima et al. 2010). Although the Survivin-Histone H3 pathway has been extensively studied, the intermolecular interactions dictating CPC-hSgo1 interactions remain unclear. My PhD work focused on characterising the molecular framework of the CPC-hSgo1 interaction using biochemical, biophysical and structural biology methods. I optimised and improved human CPC and hSgo1 recombinant protein production in an E. coli system. Post optimisation, I used Size-Exclusion Chromatography to successfully reconstitute the CPC-hSgo1 complex in vitro and further confirmed that hSgo1 possessing no modification or extra amino acids on its N-terminus can interact with Survivin and Borealin-Survivin-INCENP1-57. This suggested that the hSgo1 N-terminal tail interaction with Survivin is crucial for CPC-hSgo1 interaction. Furthermore, I conducted calorimetric binding studies to molecularly dissect the individual contributions of CPC components and their domains towards CPC-hSgo1 interaction. Towards this aim, I expressed and purified different versions of CPC and analysed their binding energetics with hSgo1. The results from these experiments clearly suggested the contribution of Borealin and INCENP towards CPC-hSgo1 interaction.

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