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Vol. 18, Issue 9, 3620-3634, September 2007

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Chlamydomonas Outer Arm Dynein Alters Conformation in Response to Ca²⁺

Miho Sakato^*,, Hitoshi Sakakibara, and Stephen M. King^*

*Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030; and Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, 588-2 Iwaoka, Nishi-ku, Kobe 651-2492, Japan

Submitted October 13, 2006; Revised July 3, 2007; Accepted July 5, 2007
Monitoring Editor: Erika Holzbaur

We have previously shown that Ca²⁺ directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the and heavy chains (HCs). The HC–associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca²⁺ with K_Ca = 3 x 10^–5 M in vitro, suggesting it may act as a Ca²⁺ sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the heavy chain. In vitro experiments indicate that LC4 undergoes a Ca²⁺-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca²⁺. The sedimentation profile of the HC subunit changed subtly upon Ca²⁺ addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca²⁺ addition. We propose that Ca²⁺-dependent conformational change of LC4 has a direct effect on the stem domain of the HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm.

¹ pCa refers to the negative log of the Ca²⁺ concentration.

² We have reanalyzed both cDNA and genomic HC clones and identified a number of differences compared to the original cDNA sequence. The amino acid numbering scheme for the HC used in this report derives from this revised sequence.

Present address: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459.

Address correspondence to: Stephen M. King (king{at}neuron.uchc.edu).

Abbreviations used: DC, docking complex; DFDNB, 1,5-difluoro-2,4-dinitrobenzene; DMP, dimethylpimelimidate; DSS, disuccinimidyl suberate; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HC, heavy chain; IC, intermediate chain; LC, light chain; UTR, untranslated region.