📄 molecular biology

Mechanism and regulation of Bim1 interactions withthe S. cerevisiae outer kinetochore Ndc80 complex

This study reveals that the microtubule plus-end tracking protein Bim1 interacts with the S. cerevisiae Ndc80 complex via a conserved SxIP motif and a secondary helical site to strengthen microtubule attachments, a process that is regulated by Ipl1/Aurora B kinase-mediated phosphorylation to facilitate the destabilization of erroneous kinetochore-microtubule connections during error correction.

Original authors: Barford, D., Winterborn, Y. B., Batters, C., Morgan, T. E., Freund, S. M.

Published 2026-08-28

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CC BY 4.0

Original authors: Barford, D., Winterborn, Y. B., Batters, C., Morgan, T. E., Freund, S. M.

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ⚕️ This is an AI-generated explanation of a preprint that has not been peer-reviewed. It is not medical advice. Do not make health decisions based on this content. Read full disclaimer

Inside every dividing cell, a microscopic machine works with relentless precision to ensure that genetic material is copied and split evenly between two new daughter cells. This machine relies on a structure called the kinetochore, a large protein complex that assembles on the chromosome and acts as a hook, grabbing onto long, thread-like fibers called microtubules. These fibers form a spindle that pulls the chromosomes apart. For this process to work without error, the kinetochore must hold on tightly enough to withstand the pulling force, yet remain flexible enough to let go if it grabs the wrong fiber. Cells have a built-in quality control system, driven by a specific enzyme, that can weaken these connections if they are not perfectly aligned, allowing the cell to try again. While scientists have long known the main parts of this hook and the fibers it grabs, they have not fully understood how other helper proteins assist in this delicate balancing act or how the cell's quality control system fine-tunes these interactions.

A team of researchers at the MRC Laboratory of Molecular Biology in Cambridge has now uncovered a previously hidden connection between two key players in this process: a protein called Bim1 and the main hooking component of the kinetochore, known as the Ndc80 complex. Bim1 is a specialized protein that naturally seeks out the growing tips of microtubules, acting like a scout that marks the end of the fiber. The Ndc80 complex is the primary structure that physically attaches the chromosome to that fiber. The researchers discovered that Bim1 does not just hang around the microtubule tip; it also reaches out and binds directly to the Ndc80 complex. This binding is not a random collision but a specific handshake mediated by a short, conserved sequence of amino acids on the Ndc80 protein. The study reveals that this interaction is strengthened by a second, less obvious contact point, creating a more stable link that helps the kinetochore hold on to the microtubule with greater force.

To find this connection, the scientists mixed purified versions of these proteins in a test tube and watched how they behaved. They observed that when Bim1 and the Ndc80 complex were combined, they stuck together, forming a larger unit that moved differently through a gel than the individual proteins did. By using a technique that measures the heat released when molecules bind, they determined that the connection is relatively weak, which is actually a feature rather than a flaw. This moderate strength allows the proteins to associate and dissociate quickly, a necessary trait for the dynamic environment of a dividing cell. The researchers then used advanced imaging and computer modeling to see exactly how they fit together. They found that the primary grip is formed by a specific motif on the Ndc80 protein, but this grip alone is not enough to explain the strength of the bond. A second region on the Ndc80 protein, which forms a small helical shape, wraps around the Bim1 protein to provide extra stability. This dual-point attachment is what allows the complex to function effectively in the cell.

The study also explored how the cell's error-correction system influences this connection. The enzyme responsible for checking for mistakes, known as Ipl1, adds chemical tags called phosphates to specific spots on the Ndc80 protein. The researchers found that when these tags are added, the shape of the Ndc80 protein changes slightly, and its ability to hold onto Bim1 weakens significantly. This suggests a clear mechanism for how the cell might loosen a faulty attachment: by modifying the Ndc80 protein, the cell reduces the grip of the helper protein Bim1, making it easier for the connection to break and reset. This finding provides a new layer of understanding to how cells ensure that chromosomes are separated correctly, preventing the genetic chaos that can lead to disease.

In a final set of experiments, the team tested whether this interaction actually helps the kinetochore hold on to the microtubule under physical stress. Using a highly sensitive optical trap that acts like a pair of microscopic tweezers, they measured the force required to pull the Ndc80 complex off a microtubule. They found that when Bim1 was present, the connection could withstand significantly more force before breaking. However, when they tested a version of the Ndc80 protein that could not bind to Bim1, the connection was weaker. This confirmed that the interaction between Bim1 and Ndc80 directly strengthens the attachment between the chromosome and the spindle fiber. Interestingly, the researchers also found that this strengthening effect does not combine with another helper protein called Dam1 to create an even stronger bond; instead, they appear to function through separate pathways or compete for the same space.

The researchers also looked at what happens inside living yeast cells to see if this interaction is essential for life. They created yeast strains where the ability of Ndc80 to bind Bim1 was disrupted, either by removing the binding site or by mimicking the phosphorylation that weakens the bond. Surprisingly, these yeast cells grew and divided normally, suggesting that while this interaction helps strengthen the connection, it is not strictly required for the cell to survive. This implies that the Bim1-Ndc80 link serves as a supportive mechanism, perhaps helping to establish the initial connection or providing extra security during the early stages of division, rather than being the sole foundation of the process. The work highlights the complexity of cellular machinery, showing that even well-studied systems like chromosome segregation rely on a network of subtle, regulated interactions that scientists are only just beginning to map.

Technical Summary: Mechanism and Regulation of the Bim1 Interaction with the Outer Kinetochore Ndc80 Complex in S. cerevisiae

Problem Statement
During eukaryotic cell division, kinetochores must couple sister chromatids to mitotic spindle microtubules to ensure faithful chromosome segregation. While the primary components of the kinetochore-microtubule interface, such as the Ndc80 complex (Ndc80c) and the Dam1 complex (Dam1c), are well-characterized, the roles of additional microtubule-associated proteins (MAPs) in facilitating efficient chromosome biorientation and segregation remain incompletely understood. Specifically, the mechanisms by which the microtubule plus-end tracking protein (+TIP) Bim1 (the budding yeast ortholog of EB1) interacts with the outer kinetochore to regulate attachment stability and error correction require further elucidation.

Methodology
The authors employed a multidisciplinary approach combining structural biology, biophysics, and cell biology to characterize the interaction between Bim1 and Ndc80c in Saccharomyces cerevisiae.

Biochemical and Biophysical Assays: Analytical size-exclusion chromatography (SEC) and SEC-MALS were used to assess complex formation and stoichiometry. Isothermal titration calorimetry (ITC) quantified binding affinities ( $K_D$ ) between Bim1 domains and various Ndc80 N-terminal (Ndc80N) constructs and peptides.
Structural Characterization: AlphaFold2 was utilized for protein structure prediction. Nuclear Magnetic Resonance (NMR) spectroscopy (HSQC, $T_2$ relaxation, secondary chemical shift analysis) provided residue-specific insights into the dynamics, secondary structure propensity, and binding interfaces of the intrinsically disordered Ndc80N region. Crosslinking mass spectrometry (CL-MS) validated proximity constraints.
Functional Assays: An optical trap rupture force assay measured the mechanical strength of Ndc80c-microtubule attachments in the presence and absence of Bim1. Total Internal Reflection Fluorescence (TIRF) microscopy visualized the recruitment of Ndc80c to dynamic microtubule plus-ends.
Genetic and In Vivo Analysis: Yeast strains with auxin-inducible degron (AID) systems were constructed to express Ndc80 mutants (e.g., SQIP motif deletions, phosphomimetic mutations) in the absence of endogenous Ndc80. Benomyl sensitivity assays assessed chromosome segregation fidelity in vivo.
Phosphorylation Studies: The error-correction kinase Ipl1 (Aurora B) was used to phosphorylate Ndc80N in vitro. Time-resolved NMR and ITC monitored the structural and affinity consequences of phosphorylation.

Key Contributions and Results

Identification of a Direct Bim1-Ndc80c Interaction:
The study establishes a previously unrecognised direct interaction between the Bim1 EBH domain and the intrinsically disordered N-terminus of Ndc80 (Ndc80N). SEC-MALS confirmed a 1:1 stoichiometry between a Bim1 dimer and the Ndc80c complex.
Dual-Binding Site Mechanism:
The interaction is mediated by two distinct sites within Ndc80N:
- Primary Site: A conserved SQIP motif (residues 22–25) within Ndc80N binds the canonical SxIP-binding pocket of the Bim1 EBH domain. Deletion of this motif or mutation of the SQIP sequence significantly reduces binding affinity.
- Secondary Site: A secondary binding site, encompassing a predicted $\alpha$ -helical segment (residues ~39–54) and C-terminal residues, engages the second SxIP-binding pocket of the Bim1 dimer. While the SQIP motif alone binds with low affinity ( $K_D \approx 178$ $\mu$ M), the inclusion of the secondary site enhances the affinity of the full Ndc80N region to the low micromolar range ( $K_D \approx 4.2$ $\mu$ M). NMR data indicate that the secondary site involves residues 42–64, with Thr54 identified as a central residue in this interaction.
Regulation by Ipl1 Phosphorylation:
Phosphorylation of Ndc80N by the error-correction kinase Ipl1 at Ser37 and Thr54 weakens the Bim1-Ndc80c interaction.
- Ipl1 phosphorylation reduces the binding affinity approximately three-fold ( $K_D$ increases from 4.2 $\mu$ M to 11.8 $\mu$ M).
- NMR analysis reveals that phosphorylation induces conformational changes, specifically increasing the flexibility of the primary binding region (residues 29–34) and destabilizing the $\alpha$ -helical propensity of the secondary binding site (residues 50–53).
- These structural perturbations, combined with electrostatic repulsion, disrupt the interaction, providing a mechanism for destabilizing incorrect kinetochore-microtubule attachments.
Functional Impact on Microtubule Attachments:
- Attachment Strength: In optical trap assays, the addition of Bim1 increased the median rupture force of Ndc80c-microtubule attachments from 5.2 pN to 8.7 pN. However, when Dam1c was present, Bim1 did not further enhance the attachment strength, suggesting that Dam1c dominates the load-bearing capacity and may compete with Ndc80c for Bim1 binding.
- Plus-End Tracking: TIRF microscopy demonstrated that Bim1 can recruit Ndc80c to dynamic microtubule plus-ends, conferring limited plus-end tracking activity to Ndc80c, which lacks this property autonomously.
- In Vivo Viability: Disruption of the SQIP motif or phosphomimetic mutations (S37E, T54E) in Ndc80 did not compromise cell viability or benomyl sensitivity in the tested yeast strains, suggesting that while the interaction is mechanistically significant, it is not strictly essential for chromosome segregation under the tested conditions, possibly due to redundancy with Dam1c-mediated mechanisms.

Significance
The paper claims to define a novel regulatory layer in kinetochore function where the +TIP protein Bim1 directly interacts with the Ndc80 complex. The authors propose that this interaction facilitates the initial recruitment and stabilization of Ndc80c at microtubule plus-ends during the establishment of end-on attachments. Crucially, the study demonstrates that the error-correction kinase Ipl1 regulates this specific interaction via phosphorylation-induced structural changes in Ndc80N. By weakening the Bim1-Ndc80c bond, Ipl1 may promote the dissociation of Ndc80c from incorrect microtubule attachments, thereby contributing to the fidelity of chromosome segregation. The findings highlight a distinct mechanism of EBH-domain recognition involving a secondary, non-canonical binding site and suggest evolutionary divergence in microtubule-coupling mechanisms between budding yeast and vertebrates, as human Ndc80N lacks the canonical motif for EB1 recognition.

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