Towards understanding of unconventional kinetochores in kinetoplastids

Thesis

The kinetochore is the macromolecular protein machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids, an evolutionarily divergent group of eukaryotes, possess a unique set of kinetochore proteins (KKT1—20, 22—25 and KKIP1—12) that lack significant similarity to known kinetochore components in other eukaryotes.

To date, KKT4 is the only component of kinetoplastid kinetochores shown to have microtubule-binding activities. However, the KKT4 structure and mechanism of action have so far remained unknown. In this thesis, I have shown that KKT4 forms oligomers and I have characterised the structure of KKT4 using X-ray crystallography, NMR spectroscopy and crosslinking mass spectrometry. I have shown that the microtubule-binding domain of KKT4 consists of a coiled-coil region followed by a positively charged disordered tail. Moreover, I demonstrated that the coiled-coil region is sufficient to interact with microtubules and the positively charged unstructured tail enhances the KKT4 affinity for microtubules. Key residues for the microtubule-binding activity have been identified using mutagenesis. I have also shown that KKT4 has DNA-binding activities in vitro.

The structure of the KKT4 C-terminal BRCT domain, revealed that the domain is a phosphorylation-dependent protein-protein interaction domain. The KKT4 BRCT domain interacts with a phosphopeptide derived from KKT8 and also makes direct contacts with the N-terminal region of microtubule-binding domain, suggesting possible regulation mechanism.

In the second part of this thesis, the structure and function of KKT23 acetyltransferase is reported. Using X-ray crystallography and NMR spectroscopy, the C-terminal region of KKT23 is shown to have a histone acetyltransferase domain which specifically acetylates histone H2A and H2AZ in vitro. The critical residues for the KKT23 catalytic activity are identified. Moreover, the N-terminal fragment of KKT23 for which no structural homolog is known, adopts a helical structure. Finally, KKT23 is shown to form a complex with KKT3 and KKT22.

Authors: Ludzia, P

• DOI: 10.5287/ora-n68oo8meq
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: KKT4; microtubules; BRCT; kinetochore; kinetoplastids; trypanosoma brucei
• Subjects: Biochemistry; Kinetochore