Tight bending of the Ndc80 complex provides intrinsic regulation of its binding to microtubules
Abstract
Regulation of the outer kinetochore complex Ndc80 is essential to ensure correct kinetochore-microtubule attachments during mitosis. Here we present a novel mechanism of regulation that is intrinsic to its structure; tight bending of the Ndc80 complex inhibits its microtubule binding. Using single molecule Förster resonance energy transfer (FRET), we show that the S. cerevisiae Ndc80 complex can fluctuate between straight and bent forms, and that binding of the complex to microtubules selects for straightened forms. The loop region of the complex enables its bent conformation, as deletion of the loop promotes straightening. In addition, the kinetochore complex MIND enhances microtubule binding by opposing the tightly bent, auto-inhibited conformation of the Ndc80 complex. We suggest that prior to its assembly at the kinetochore, the Ndc80 complex interchanges between bent (auto-inhibited) and open conformations. Once assembled, its association with MIND stabilizes the Ndc80 complex in a straightened form for higher affinity microtubule binding.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1, 2, 3, 4 and 5, as well as Figure 1-figure supplement 1, Figure 3-figure supplement 1, Figure 4-figure supplement 1 and Figure 5-figure supplement 1.
Article and author information
Author details
Funding
National Institute of General Medical Sciences (T32 GM007270)
- Emily Anne Scarborough
National Institute of General Medical Sciences (R01 GM040506)
- Trisha N Davis
National Institute of General Medical Sciences (R01 GM079373)
- Charles L Asbury
National Institute of General Medical Sciences (P01 GM105537)
- Charles L Asbury
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2019, Scarborough et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Further reading
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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for head–head coordination; however, which kinetic step(s) in the chemomechanical cycle is ‘gated’ by the neck linker remains unresolved. Here, we employed pre-steady-state kinetics and single-molecule assays to investigate how the neck-linker conformation affects kinesin’s motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubule, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.
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- Biochemistry and Chemical Biology
- Computational and Systems Biology
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