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. It 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 a spindle checkpoint protein Bub1. In addition, KKT14 has a putative ABBA motif that is present in Bub1 and its paralogue 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 orthologues of Bub1/BubR1-Bub3, which promote accurate chromosome segregation in trypanosomes.

NMR study of the structure and dynamics of the BRCT domain from the kinetochore protein KKT4.

KKT4 is a multi-domain kinetochore protein specific to kinetoplastids, such as Trypanosoma brucei. It lacks significant sequence similarity to known kinetochore proteins in other eukaryotes. Our recent X-ray structure of the C-terminal region of KKT4 shows that it has a tandem BRCT (BRCA1 C Terminus) domain fold with a sulfate ion bound in a typical binding site for a phosphorylated serine or threonine. Here we present the 1H, 13C and 15N resonance assignments for the BRCT domain of KKT4 (KKT4463-645) from T. brucei. We show that the BRCT domain can bind phosphate ions in solution using residues involved in sulfate ion binding in the X-ray structure. We have used these assignments to characterise the secondary structure and backbone dynamics of the BRCT domain in solution. Mutating the residues involved in phosphate ion binding in T. brucei KKT4 BRCT results in growth defects confirming the importance of the BRCT phosphopeptide-binding activity in vivo. These results may facilitate rational drug design efforts in the future to combat diseases caused by kinetoplastid parasites.

Divergent polo boxes in KKT2 bind KKT1 to 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. Here we characterize two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres. They 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 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 trypanosomes. Furthermore, we demonstrate that the divergent polo boxes of KKT2 interact directly with KKT1 and that KKT1 interacts with KKT6. These results show that the divergent polo boxes of KKT2 and KKT3 are protein-protein interaction domains that initiate kinetochore assembly in T. brucei.

Repurposing of synaptonemal complex proteins for kinetochores in Kinetoplastida.

Chromosome segregation in eukaryotes is driven by the kinetochore, a macromolecular complex that connects centromeric DNA to microtubules of the spindle apparatus. Kinetochores in well-studied model eukaryotes consist of a core set of proteins that are broadly conserved among distant eukaryotic phyla. By contrast, unicellular flagellates of the class Kinetoplastida have a unique set of 36 kinetochore components. The evolutionary origin and history of these kinetochores remain unknown. Here, we report evidence of homology between axial element components of the synaptonemal complex and three kinetoplastid kinetochore proteins KKT16-18. The synaptonemal complex is a zipper-like structure that assembles between homologous chromosomes during meiosis to promote recombination. By using sensitive homology detection protocols, we identify divergent orthologues of KKT16-18 in most eukaryotic supergroups, including experimentally established chromosomal axis components, such as Red1 and Rec10 in budding and fission yeast, ASY3-4 in plants and SYCP2-3 in vertebrates. Furthermore, we found 12 recurrent duplications within this ancient eukaryotic SYCP2-3 gene family, providing opportunities for new functional complexes to arise, including KKT16-18 in the kinetoplastid parasite Trypanosoma brucei. We propose the kinetoplastid kinetochore system evolved by repurposing meiotic components of the chromosome synapsis and homologous recombination machinery that were already present in early eukaryotes.

Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein.

The 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.

1H, 13C and 15N resonance assignments for the microtubule-binding domain of the kinetoplastid kinetochore protein KKT4 from Trypanosoma brucei.

KKT4 is a kinetoplastid-specific microtubule-binding kinetochore protein that lacks significant similarity to any known kinetochore or microtubule-binding proteins. Here we present the 1H, 13C and 15N resonance assignments for several fragments from the microtubule-binding domain of KKT4 (KKT4115-343) from Trypanosoma brucei. These assignments provide the starting point for detailed investigations of the structure, dynamics and interactions of the microtubule-binding region of KKT4.

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, 12 proteins (KKT-interacting proteins 1-12, 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 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.

The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip-coupling protein.

Kinetochores are multiprotein machines that drive chromosome segregation by maintaining persistent, load-bearing linkages between chromosomes and dynamic microtubule tips. Kinetochores in commonly studied eukaryotes bind microtubules through widely conserved components like the Ndc80 complex. 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 the T. brucei kinetochore protein KKT4 binds directly to microtubules and maintains load-bearing attachments to both growing and shortening 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 microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Thus, despite its lack of significant similarity to other known microtubule-binding proteins, KKT4 has key functions required for driving chromosome segregation. We propose that it represents a primary element of the kinetochore-microtubule interface in kinetoplastids.

Design and In Vitro Evaluation of a Cytotoxic Conjugate Based on the Anti-HER2 Affibody Fused to the Fc Fragment of IgG1.

In our previous work we demonstrated that a small protein called affibody can be used for a cytotoxic conjugate development. The anti-HER2 affibody was armed with one moiety of a highly potent auristatin E and specifically killed HER2-positive cancer cells with a nanomolar IC50. The aim of this study was to improve the anti-HER2 affibody conjugate by increasing its size and the number of conjugated auristatin molecules. The affibody was fused to the Fc fragment of IgG1 resulting in a dimeric construct with the molecular weight of 68 kDa, referred to as ZHER2:2891-Fc, ensuring its prolonged half-life in the blood. Due to the presence of four interchain cysteines, the fusion protein could carry four drug molecules. Notably, the in vitro tests of the improved anti-HER2 conjugate revealed that it exhibits the IC50 of 130 pM for the HER2-positive SK-BR-3 cells and 98 nM for the HER2-negative MDA-MB-231 cells. High efficacy and specificity of the auristatin conjugate based on ZHER2:2891-Fc indicate that this construct is suitable for further in vivo evaluation.