Insights into dynein motor domain function from a 3.3-Å crystal structure

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Abstract

Dyneins power the beating of cilia and flagella, transport various intracellular cargos and are necessary for mitosis. All dyneins have a 300-kDa motor domain consisting of a ring of six AAA+ domains. ATP hydrolysis in the AAA+ ring drives the cyclic relocation of a motile element, the linker domain, to generate the force necessary for movement. How the linker interacts with the ring during the ATP hydrolysis cycle is not known. Here we present a 3.3-Å crystal structure of the motor domain of Saccharomyces cerevisiae cytoplasmic dynein, crystallized in the absence of nucleotides. The linker is docked to a conserved site on AAA5, which is confirmed by mutagenesis as functionally necessary. Nucleotide soaking experiments show that the main ATP hydrolysis site in dynein (AAA1) is in a low-nucleotide affinity conformation and reveal the nucleotide interactions of the other three sites (AAA2, AAA3 and AAA4).

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Figure 1: Overview of the dynein motor domain.
Figure 2: Interaction of the linker with the AAA+ ring.
Figure 3: Conservation plots of the dynein motor domain.
Figure 4: Nucleotide binding sites in the AAA+ ring.
Figure 5: Schematic representation of the nucleotide-free dynein motor domain.

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Acknowledgements

We thank C. Cho for her work helping to identify heavy atom derivatives and suitable crystallization conditions. We also thank M. Schlager, A. Diamant, C. Cho, R. Vale, K. Nagai and L. Passmore for helpful discussions and their comments on the manuscript. This work was supported by the Medical Research Council (MC_UP_A025_1011 to A.P.C.).

Author information

E.S.G., H.S. and A.P.C. produced, purified and crystallized the protein. H.S. and E.S.G. prepared heavy atom derivatives. H.S. and A.P.C. collected data on crystals and determined the structure. H.S. carried out phasing. All authors built the model. E.S.G. and A.P.C. conducted the in vitro experiments. A.P.C. and H.S. wrote the paper.

Correspondence to Andrew P Carter.

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