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

AbstractThe 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.

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Faculty Opinions recommendation of A microtubule-binding domain in dynactin increases dynein processivity by skating along microtubules.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1030972.363474 ◽

2006 ◽

Author(s):

David Stephens

Keyword(s):

Binding Domain ◽

Microtubule Binding ◽

Microtubule Binding Domain

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Faculty Opinions recommendation of Impacts of Cd(II) on the conformation and self-aggregation of Alzheimer's tau fragment corresponding to the third repeat of microtubule-binding domain.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.719611760.793515201 ◽

2016 ◽

Author(s):

Michael Aschner

Keyword(s):

Binding Domain ◽

Microtubule Binding ◽

The Third ◽

Microtubule Binding Domain ◽

Self Aggregation

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Structure and Functional Role of Dynein's Microtubule-Binding Domain

Science ◽

10.1126/science.1164424 ◽

2008 ◽

Vol 322 (5908) ◽

pp. 1691-1695 ◽

Cited By ~ 172

Author(s):

A. P. Carter ◽

J. E. Garbarino ◽

E. M. Wilson-Kubalek ◽

W. E. Shipley ◽

C. Cho ◽

...

Keyword(s):

Functional Role ◽

Binding Domain ◽

Microtubule Binding ◽

Microtubule Binding Domain

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The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip–coupling protein

The Journal of Cell Biology ◽

10.1083/jcb.201711181 ◽

2018 ◽

Vol 217 (11) ◽

pp. 3886-3900 ◽

Cited By ~ 12

Author(s):

Aida Llauró ◽

Hanako Hayashi ◽

Megan E. Bailey ◽

Alex Wilson ◽

Patryk Ludzia ◽

...

Keyword(s):

Chromosome Segregation ◽

Binding Activity ◽

Significant Similarity ◽

Load Bearing ◽

Kinetochore Protein ◽

Microtubule Binding ◽

Binding Domains ◽

Microtubule Binding Domain ◽

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.

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Autoregulatory control of microtubule binding in doublecortin-like kinase 1

eLife ◽

10.7554/elife.60126 ◽

2021 ◽

Vol 10 ◽

Author(s):

Regina L Agulto ◽

Melissa M Rogers ◽

Tracy C Tan ◽

Amrita Ramkumar ◽

Ashlyn M Downing ◽

...

Keyword(s):

Binding Affinity ◽

Therapeutic Target ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Binding Domain ◽

Cancer Development ◽

Microtubule Binding ◽

Microtubule Binding Domain

The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We find that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 have diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.

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The projection domain of MAP2b regulates microtubule protrusion and process formation in Sf9 cells

Journal of Cell Science ◽

10.1242/jcs.115.7.1523 ◽

2002 ◽

Vol 115 (7) ◽

pp. 1523-1539 ◽

Cited By ~ 1

Author(s):

Dave Bélanger ◽

Carole Abi Farah ◽

Minh Dang Nguyen ◽

Michel Lauzon ◽

Sylvie Cornibert ◽

...

Keyword(s):

Cell Surface ◽

Binding Domain ◽

Intramolecular Interactions ◽

Microtubule Assembly ◽

Sf9 Cells ◽

Microtubule Binding ◽

Process Formation ◽

Microtubule Binding Domain ◽

Projection Domain

The expression of microtubule-associated protein 2 (MAP2), developmentally regulated by alternative splicing, coincides with neurite outgrowth. MAP2 proteins contain a microtubule-binding domain (C-terminal) that promotes microtubule assembly and a poorly characterized domain, the projection domain(N-terminal), extending at the surface of microtubules. MAP2b differs from MAP2c by an additional sequence of 1372 amino acids in the projection domain. In this study, we examined the role of the projection domain in the protrusion of microtubules from the cell surface and the subsequent process formation in Sf9 cells. In this system, MAP2b has a lower capacity to induce process formation than MAP2c. To investigate the role of the projection domain in this event, we expressed truncated forms of MAP2b and MAP2c that have partial or complete deletion of their projection domain in Sf9 cells. Our results indicate that process formation is induced by the microtubule-binding domain of these MAP2 proteins and is regulated by their projection domain. Furthermore, the microtubule-binding activity of MAP2b and MAP2c truncated forms as well as the structural properties of the microtubule bundles induced by them do not seem to be the only determinants that control the protrusion of microtubules from the cell surface in Sf9 cells. Rather, our data suggest that microtubule protrusion and process formation are regulated by intramolecular interactions between the projection domain and its microtubule-binding domain in MAP2b.

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