Abstract
SummaryThe kinetochore is the macromolecular machinery that drives chromosome segregation by interacting with spindle microtubules. Kinetoplastids (such as Trypanosoma brucei), a group of evolutionarily divergent eukaryotes, have a unique set of kinetochore proteins that lack any significant homology to canonical kinetochore components. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterize the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Taken together, these results provide structural insights into the unconventional kinetoplastid kinetochore protein KKT4.
Highlights
Every time a cell divides, it must duplicate and segregate its genetic material accurately into two daughter cells
To determine the oligomerization state of KKT4, we used size-exclusion chromatography coupled with multi-angle light scattering (SECMALS) (Wen et al, 1996) (Figure S1)
The following predicted structural regions of KKT4 are well conserved among kinetoplastids: N-terminal a helix, coiled coil and block of basic residues within the microtubule-binding domain, and the C-terminal breast cancer-associated protein 1 (BRCA1) C-terminal (BRCT) domain (Figures 1 and S2) (Llauro et al, 2018)
Summary
Every time a cell divides, it must duplicate and segregate its genetic material accurately into two daughter cells. A key structure involved in chromosome segregation in eukaryotes is the kinetochore, a macromolecular protein complex that assembles onto centromeric DNA and interacts with spindle microtubules during mitosis and meiosis (Mcintosh, 2016). Microtubules are dynamic polymers that change in length by addition or removal of tubulin subunits at the tips (Desai and Mitchison, 1997). Accurate chromosome segregation requires that kinetochores form robust attachments to the dynamic microtubule tips. Kinetochores need to destabilize erroneous attachments to ensure that sister kinetochores bind microtubules emanating from opposite poles (Nicklas, 1997; Biggins, 2013; Cheeseman, 2014; Musacchio and Desai, 2017). Revealing the molecular basis of kinetochore-microtubule attachments and their regulation is key to understanding the mechanism of chromosome segregation
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