Biology subjects

Ludzia, P.

Publications and source records attributed to Ludzia, P..

7 recordsLinked to original sources

bioRxiv2026

The centromere localization domain of kinetoplastid kinetochore protein KKT2 recognizes the free N-terminus of histone H3

Kinetochores are multiprotein complexes that drive chromosome segregation in eukaryotes. Kinetoplastids, a group of early-diverging eukaryotes that lack canonical kinetochore components, have a unique set of kinetochore proteins. How their kinetochores are assembled specifically at centromeres in the absence of a centromere-specific histone H3 variant CENP-A remains unknown. Here, we demonstrate that the centromere localization (CL) domain of KKT2 has similarities to a ZZ domain. An invariant aspartate present in histone H3-binding ZZ domains is conserved in the KKT2 CL domain. Using nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we show that Trypanosoma brucei KKT2 CL binds the N-terminus of histone H3. Strikingly, even mono-methylation of the N-terminal amino group of histone H3 abolishes the binding. Our study raises a possibility that centromere-specific kinetochore assembly in T. brucei is ensured by abundant N-terminal methylations of histone H3 in non-centromeric regions.

cell biology↗

bioRxiv2024

The kinetoplastid kinetochore protein KKT23 acetyltransferase is a structural homolog of GCN5 that acetylates the histone H2A C-terminal tail

The kinetochore is the macromolecular protein machine that drives chromosome segregation in eukaryotes. In an evolutionarily divergent group of organisms called kinetoplastids, kinetochores are built using a unique set of proteins (KKT1-25 and KKIP1-12). KKT23 is a constitutively localised kinetochore protein containing a C-terminal acetyltransferase domain of unknown function. Here, using X-ray crystallography and NMR spectroscopy, we have determined the structure and dynamics of the KKT23 acetyltransferase domain from Trypanosoma brucei and found that it is structurally similar to the GCN5 histone acetyltransferase domain. We find that KKT23 can acetylate the C-terminal tail of histone H2A and that knockdown of KKT23 results in decreased H2A acetylation levels in T. brucei. Finally, we have determined the crystal structure of the N-terminal region of KKT23 and shown that it interacts with KKT22. Our study provides important insights into the structure and function of the unique kinetochore acetyltransferase in trypanosomes.

cell biology↗

bioRxiv2024

Kinetoplastid kinetochore proteins KKT14-KKT15 are divergent Bub1/BubR1-Bub3 proteins

Faithful transmission of genetic material is crucial for the survival of all organisms. In many eukaryotes, a feedback control mechanism called the spindle checkpoint ensures chromosome segregation fidelity by delaying cell cycle progression until all chromosomes achieve proper attachment to the mitotic spindle. Kinetochores are the macromolecular complexes that act as the interface between chromosomes and spindle microtubules. While most eukaryotes have canonical kinetochore proteins that are widely conserved, kinetoplastids such as Trypanosoma brucei have a seemingly unique set of kinetochore proteins including KKT1-25. Available evidence suggests that T. brucei lacks components of the spindle checkpoint and is unable to delay cell cycle progression in response to spindle defects. It therefore remains poorly understood how kinetoplastids regulate cell cycle progression or ensure chromosome segregation fidelity. Here, we report a crystal structure of the C-terminal domain of KKT14 from Apiculatamorpha spiralis and uncover that it is a pseudokinase. Its structure is most similar to the kinase domain of Bub1, a key component of the spindle checkpoint in other eukaryotes. In addition, KKT14 in T. brucei and other trypanosomatids has a putative ABBA motif that is present in Bub1 and its paralog BubR1. We also find that the N-terminal part of KKT14 interacts with KKT15, whose WD40 repeat beta-propeller is phylogenetically closely related to a direct interactor of Bub1/BubR1 called Bub3. Our findings indicate that KKT14-KKT15 are divergent orthologs of Bub1/BubR1-Bub3, which promote accurate chromosome segregation in trypanosomes.

cell biology↗

bioRxiv2022

Divergent polo boxes in KKT2 and KKT3 initiate the kinetochore assembly cascade in Trypanosoma brucei

Chromosome segregation requires assembly of the macromolecular kinetochore complex onto centromeric DNA. While most eukaryotes have canonical kinetochore proteins that are widely conserved among eukaryotes, evolutionarily divergent kinetoplastids have a unique set of kinetochore proteins. Little is known about the mechanism of kinetochore assembly in kinetoplastids. In this study, we have characterized two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres and promote the recruitment of other kinetochore proteins. KKT2 and KKT3 have three domains that are highly conserved among kinetoplastids: an N-terminal kinase domain of unknown function, the centromere localization domain in the middle, and the C-terminal domain that has weak similarity to polo boxes of Polo-like kinases. We show that the kinase activity of KKT2 is essential for accurate chromosome segregation, while that of KKT3 is dispensable for cell growth in the procyclic form of Trypanosoma brucei. Crystal structures of their divergent polo boxes reveal differences between KKT2 and KKT3. We also show that the divergent polo boxes of KKT3 are sufficient to recruit KKT2 in T. brucei. Furthermore, we identify the C-terminal domain of KKT1 as a direct interaction partner for the divergent polo boxes of KKT2, but not KKT3. KKT6 is found to interact with KKT1. These results show that KKT2 and KKT3 recruit downstream kinetochore proteins by direct protein-protein interactions using their divergent polo boxes.

cell biology↗

bioRxiv2020

Structural characterisation of KKT4, an unconventional microtubule-binding kinetochore protein

The kinetochore is the macromolecular protein machinery that drives chromosome segregation by interacting with spindle microtubules. Unlike most other eukaryotes that have canonical kinetochore proteins, a group of evolutionarily divergent eukaryotes called kinetoplastids (such as Trypanosoma brucei) have a unique set of kinetochore proteins. 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 characterise 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 crystal 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. Finally, we show that KKT4 binds DNA with high affinity. Taken together, these results provide the first structural insights into the unconventional kinetoplastid kinetochore protein KKT4.

cell biology↗

bioRxiv2019

Identification of four unconventional kinetoplastid kinetochore proteins KKT22-25 in Trypanosoma brucei

The kinetochore is a multi-protein complex that drives chromosome segregation in eukaryotes. It assembles onto centromere DNA and interacts with spindle microtubules during mitosis and meiosis. Although most eukaryotes have canonical kinetochore proteins, kinetochores of evolutionarily divergent kinetoplastid species consist of at least 20 unconventional kinetochore proteins (KKT1-20). In addition, twelve proteins (KKIP1-12) are known to localize at kinetochore regions during mitosis. It remains unclear whether KKIP proteins interact with KKT proteins. Here, we report the identification of four additional kinetochore proteins, KKT22-25, in Trypanosoma brucei. KKT22 and KKT23 constitutively localize at kinetochores, while KKT24 and KKT25 localize from S phase to anaphase. KKT23 has a Gcn5-related N-acetyltransferase (GNAT) domain, which is not found in any kinetochore protein known to date. We also show that KKIP1 co-purifies with KKT proteins, but not with KKIP proteins. Finally, our affinity purification of KKIP2/3/4/6 identifies a number of proteins as their potential interaction partners, many of which are implicated in RNA binding or processing. These findings further support the idea that kinetoplastid kinetochores are unconventional.

cell biology↗

bioRxiv2017

The unconventional kinetoplastid kinetochore protein KKT4 tracks with dynamic microtubule tips

Kinetochores are multiprotein machines that drive chromosome segregation in all eukaryotes by maintaining persistent, load-bearing linkages between the chromosomes and the tips of dynamic spindle microtubules. Kinetochores in commonly studied eukaryotes are assembled from widely conserved components like the Ndc80 complex that directly binds microtubules. However, in evolutionarily-divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here we show that a kinetochore protein from T. brucei called KKT4 binds directly to microtubules, diffuses along the microtubule lattice, and tracks with disassembling microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a minimal microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Laser trapping experiments show that KKT4 can maintain load-bearing attachments to both growing and shortening microtubule tips. Thus, despite its lack of similarity to other known microtubule-binding proteins, KKT4 has key functions required for harnessing microtubule dynamics to drive chromosome segregation. We propose that it represents a primary element of the kinetochore-microtubule interface in kinetoplastids.

cell biology↗