Members of the dynein family, consisting of cytoplasmic and axonemal isoforms, are motors that move towards the minus ends of microtubules. Cytoplasmic dynein-1 (dynein-1) plays roles in mitosis and cellular cargo transport 1 , and is implicated in viral infections 2 and neurodegenerative diseases 3 . Cytoplasmic dynein-2 (dynein-2) carries out intraflagellar transport 4 and is associated with human skeletal ciliopathies 5 . Dyneins share a conserved motor domain that couples cycles of ATP hydrolysis with conformational changes to produce movement 6-9 . Here we present the crystal structure of the human cytoplasmic dynein-2 motor bound to the ATP-hydrolysis transition state analogue ADP.vanadate (ADP.Vi) 10 . The structure reveals a closure of the motor's ring of six AAA+ domains (ATPases associated with various cellular activites: AAA1-AAA6). This induces a steric clash with the linker, the key element for the generation of movement, driving it into a conformation that is primed to produce force. Ring closure also changes the interface between the stalk and buttress coiled-coil extensions of the motor domain. This drives helix sliding in the stalk that causes the microtubule binding domain (MTBD) at its tip to release from the microtubule. Our structure answers the key questions of how ATP hydrolysis leads to linker remodelling and microtubule affinity regulation.There are four nucleotide-binding sites in the dynein motor, but movement only depends on ATP hydrolysis in the first site (AAA1) 7,11,12 . When this site is nucleotide free or bound to ADP, the MTBD binds to the microtubule and the linker adopts the straight postpowerstroke conformation [6][7][8][12][13][14] . Upon ATP binding and hydrolysis, the MTBD detaches from the microtubule and the linker is primed into the pre-powerstroke

SummaryDyneins power the beating of cilia and flagella, transport various intracellular cargos and are important during mitosis. All dyneins have a ~300kDa 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 3.3Å 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, confirmed by mutagenesis as functionally important. 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 (AAA2-4).

Dyneins are multiprotein complexes that move cargo along microtubules in the minus end direction. The largest individual component of the dynein complex is the heavy chain. Its C‐terminal 3500 amino‐acid residues form the motor domain, which hydrolyses ATP in its ring of AAA+ (ATPases associated with diverse cellular activities) domains to generate the force for movement. The production of force is synchronized with cycles of microtubule binding and release, another important prerequisite for efficient motility along the microtubule. Although the large scale conformational changes that lead to force production and microtubule affinity regulation are well established, it has been largely enigmatic how ATP‐hydrolysis in the AAA+ ring causes these rearrangements. The past five years have seen a surge of high resolution information on the dynein motor domain that finally allowed unprecedented insights into this important open question. This review, part of the “ATP and GTP hydrolysis in Biology” special issue, will summarize our current understanding of the dynein motor mechanism with a special emphasis on the recently obtained crystal and EM structures. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 557–567, 2016.