The complex of outer-arm dynein light chain-1 and the microtubule-binding domain of the γ heavy chain shows how axonemal dynein tunes ciliary beating

Axonemal dynein is a microtubule-based molecular motor that drives ciliary/flagellar beating in eukaryotes. In axonemal dynein, the outer-arm dynein (OAD) complex, which comprises three heavy chains (α, β, and γ), produces the main driving force for ciliary/flagellar motility. It has recently been shown that axonemal dynein light chain-1 (LC1) binds to the microtubule-binding domain (MTBD) of OADγ, leading to a decrease in its microtubule-binding affinity. However, it remains unclear how LC1 interacts with the MTBD and controls the microtubule-binding affinity of OADγ. Here, we have used X-ray crystallography and pulldown assays to examine the interaction between LC1 and the MTBD, identifying two important sites of interaction in the MTBD. Solving the LC1-MTBD complex from Chlamydomonas reinhardtii at 1.7 Å resolution, we observed that one site is located in the H5 helix and that the other is located in the flap region that is unique to some axonemal dynein MTBDs. Mutational analysis of key residues in these sites indicated that the H5 helix is the main LC1-binding site. We modeled the ternary structure of the LC1-MTBD complex bound to microtubules based on the known dynein-microtubule complex. This enabled us to propose a structural basis for both formations of the ternary LC1-MTBD-microtubule complex and LC1-mediated tuning of MTBD binding to the microtubule, suggesting a molecular model for how axonemal dynein senses the curvature of the axoneme and tunes ciliary/flagellar beating.

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Axonemal dynein light chain-1 locates at the microtubule-binding domain of the γ heavy chain

Molecular Biology of the Cell ◽

10.1091/mbc.e15-05-0289 ◽

2015 ◽

Vol 26 (23) ◽

pp. 4236-4247 ◽

Cited By ~ 14

Author(s):

Muneyoshi Ichikawa ◽

Kei Saito ◽

Haru-aki Yanagisawa ◽

Toshiki Yagi ◽

Ritsu Kamiya ◽

...

Keyword(s):

Light Chain ◽

Regulatory Mechanism ◽

Binding Domain ◽

Particle Analysis ◽

Dynein Light Chain ◽

Microtubule Binding ◽

Heavy Chains ◽

Microtubule Binding Domain ◽

Axonemal Dynein ◽

Force Generator

The outer arm dynein (OAD) complex is the main propulsive force generator for ciliary/flagellar beating. In Chlamydomonas and Tetrahymena, the OAD complex comprises three heavy chains (α, β, and γ HCs) and >10 smaller subunits. Dynein light chain-1 (LC1) is an essential component of OAD. It is known to associate with the Chlamydomonas γ head domain, but its precise localization within the γ head and regulatory mechanism of the OAD complex remain unclear. Here Ni-NTA-nanogold labeling electron microscopy localized LC1 to the stalk tip of the γ head. Single-particle analysis detected an additional structure, most likely corresponding to LC1, near the microtubule-binding domain (MTBD), located at the stalk tip. Pull-down assays confirmed that LC1 bound specifically to the γ MTBD region. Together with observations that LC1 decreased the affinity of the γ MTBD for microtubules, we present a new model in which LC1 regulates OAD activity by modulating γ MTBD's affinity for the doublet microtubule.

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Faculty Opinions recommendation of Axonemal dynein light chain-1 locates at the microtubule-binding domain of the γ heavy chain.

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

10.3410/f.725807212.793510822 ◽

2015 ◽

Author(s):

Winfield S Sale ◽

Emily Hunter

Keyword(s):

Heavy Chain ◽

Light Chain ◽

Binding Domain ◽

Dynein Light Chain ◽

Microtubule Binding ◽

Microtubule Binding Domain ◽

Axonemal Dynein

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The Complex of Outer-arm Dynein Light Chain-1 and the Microtubule-binding Domain of the Heavy Chain Shows How Axonemal Dynein Tunes Ciliary Beating

Seibutsu Butsuri ◽

10.2142/biophys.61.020 ◽

2021 ◽

Vol 61 (1) ◽

pp. 020-022

Author(s):

Toshiki YAGI ◽

Akiyuki TODA ◽

Muneyoshi ICHIKAWA ◽

Genji KURISU

Keyword(s):

Heavy Chain ◽

Light Chain ◽

Binding Domain ◽

Dynein Light Chain ◽

Microtubule Binding ◽

Ciliary Beating ◽

Microtubule Binding Domain ◽

Axonemal Dynein

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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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