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An outer arm dynein light chain acts in a conformational switch for flagellar motility.

Patel-King RS, King SM - J. Cell Biol. (2009)

Bottom Line: Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme.Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain.We propose that this gamma HC-LC1-microtubule ternary complex functions as a conformational switch to control outer arm activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
A system distinct from the central pair-radial spoke complex was proposed to control outer arm dynein function in response to alterations in the mechanical state of the flagellum. In this study, we examine the role of a Chlamydomonas reinhardtii outer arm dynein light chain that associates with the motor domain of the gamma heavy chain (HC). We demonstrate that expression of mutant forms of LC1 yield dominant-negative effects on swimming velocity, as the flagella continually beat out of phase and stall near or at the power/recovery stroke switchpoint. Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme. Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain. We propose that this gamma HC-LC1-microtubule ternary complex functions as a conformational switch to control outer arm activity.

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Structure-based design of LC1 Mutants. The mean LC1 ribbon structure (Protein Data Bank accession no. 1M9L; left) and three views of the van der Waals molecular surface (right) are shown. The LRR region forms two β sheets and a helical face. The larger β sheet face (left, magenta) contains a single hydrophobic patch (green) centered on W99 that is predicted to bind the γ HC. The C-terminal portion of LC1 consists of a helical region, the orientation of which is controlled by two residues (M182 [yellow] and D185 [blue]) that show high backbone dynamics. The terminal α9 helix likely protrudes into the AAA+ domain and contains two Arg residues (R189 and R196; pink) that potentially make ionic contacts with the motor domain and/or nucleotide.
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fig1: Structure-based design of LC1 Mutants. The mean LC1 ribbon structure (Protein Data Bank accession no. 1M9L; left) and three views of the van der Waals molecular surface (right) are shown. The LRR region forms two β sheets and a helical face. The larger β sheet face (left, magenta) contains a single hydrophobic patch (green) centered on W99 that is predicted to bind the γ HC. The C-terminal portion of LC1 consists of a helical region, the orientation of which is controlled by two residues (M182 [yellow] and D185 [blue]) that show high backbone dynamics. The terminal α9 helix likely protrudes into the AAA+ domain and contains two Arg residues (R189 and R196; pink) that potentially make ionic contacts with the motor domain and/or nucleotide.

Mentions: LC1 consists of an N-terminal helix capping a ββα barrel and a C-terminal helical region that protrudes from the main protein axis (Fig. 1; Wu et al., 1999, 2000, 2003). The hydrophobic patch (Fig. 1, colored green on the molecular surface) centered on W99 within the large β sheet is thought to mediate association with the γ HC, as this interaction is stable to high salt (Wu et al., 2000). Consequently, we predicted that the C-terminal α9 helix likely protrudes into the HC AAA+ domains. Previously, we demonstrated that two residues, M182 (Fig. 1, yellow) and D185 (Fig. 1, blue), exhibit high backbone dynamics (Wu et al., 2003) and may control the orientation of this helix. Furthermore, the α9 helix contains two Arg residues (R189 and R196; Fig. 1, pink) that potentially make ionic contacts within the AAA+ domains or with bound nucleotides (Wu et al., 2000, 2003); these latter residues were targeted, as, unlike the α and β HCs, the γ HC exhibits maximal ATPase activity at pH 10.0, suggesting that the ionization state of a basic residue is important in the catalytic and/or regulatory mechanism (Gatti et al., 1991).


An outer arm dynein light chain acts in a conformational switch for flagellar motility.

Patel-King RS, King SM - J. Cell Biol. (2009)

Structure-based design of LC1 Mutants. The mean LC1 ribbon structure (Protein Data Bank accession no. 1M9L; left) and three views of the van der Waals molecular surface (right) are shown. The LRR region forms two β sheets and a helical face. The larger β sheet face (left, magenta) contains a single hydrophobic patch (green) centered on W99 that is predicted to bind the γ HC. The C-terminal portion of LC1 consists of a helical region, the orientation of which is controlled by two residues (M182 [yellow] and D185 [blue]) that show high backbone dynamics. The terminal α9 helix likely protrudes into the AAA+ domain and contains two Arg residues (R189 and R196; pink) that potentially make ionic contacts with the motor domain and/or nucleotide.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2717645&req=5

fig1: Structure-based design of LC1 Mutants. The mean LC1 ribbon structure (Protein Data Bank accession no. 1M9L; left) and three views of the van der Waals molecular surface (right) are shown. The LRR region forms two β sheets and a helical face. The larger β sheet face (left, magenta) contains a single hydrophobic patch (green) centered on W99 that is predicted to bind the γ HC. The C-terminal portion of LC1 consists of a helical region, the orientation of which is controlled by two residues (M182 [yellow] and D185 [blue]) that show high backbone dynamics. The terminal α9 helix likely protrudes into the AAA+ domain and contains two Arg residues (R189 and R196; pink) that potentially make ionic contacts with the motor domain and/or nucleotide.
Mentions: LC1 consists of an N-terminal helix capping a ββα barrel and a C-terminal helical region that protrudes from the main protein axis (Fig. 1; Wu et al., 1999, 2000, 2003). The hydrophobic patch (Fig. 1, colored green on the molecular surface) centered on W99 within the large β sheet is thought to mediate association with the γ HC, as this interaction is stable to high salt (Wu et al., 2000). Consequently, we predicted that the C-terminal α9 helix likely protrudes into the HC AAA+ domains. Previously, we demonstrated that two residues, M182 (Fig. 1, yellow) and D185 (Fig. 1, blue), exhibit high backbone dynamics (Wu et al., 2003) and may control the orientation of this helix. Furthermore, the α9 helix contains two Arg residues (R189 and R196; Fig. 1, pink) that potentially make ionic contacts within the AAA+ domains or with bound nucleotides (Wu et al., 2000, 2003); these latter residues were targeted, as, unlike the α and β HCs, the γ HC exhibits maximal ATPase activity at pH 10.0, suggesting that the ionization state of a basic residue is important in the catalytic and/or regulatory mechanism (Gatti et al., 1991).

Bottom Line: Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme.Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain.We propose that this gamma HC-LC1-microtubule ternary complex functions as a conformational switch to control outer arm activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.

ABSTRACT
A system distinct from the central pair-radial spoke complex was proposed to control outer arm dynein function in response to alterations in the mechanical state of the flagellum. In this study, we examine the role of a Chlamydomonas reinhardtii outer arm dynein light chain that associates with the motor domain of the gamma heavy chain (HC). We demonstrate that expression of mutant forms of LC1 yield dominant-negative effects on swimming velocity, as the flagella continually beat out of phase and stall near or at the power/recovery stroke switchpoint. Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme. Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain. We propose that this gamma HC-LC1-microtubule ternary complex functions as a conformational switch to control outer arm activity.

Show MeSH
Related in: MedlinePlus