Multiple nucleotide-binding sites in the sequence of dynein β heavy chain

Overlapping cDNA clones encoding the heavy chain of rat brain cytoplasmic dynein have been isolated. The isolated cDNA clones contain an open reading frame of 13,932 bp encoding 4644 aa (Mr, 532,213). The deduced protein sequence of the heavy chain of rat brain dynein shows significant similarity to sea urchin flageilar -dynein (27.0% identical) and toDictyostelium cytoplasmic dynein (53.5% identical) throughout the entire sequence. The heavy chain of rat brain (cytoplasmic) dynein contains four putative nudeotide-binding consensus sequences [GX4GK(T/S)] in the central one-third region that are highly similar to those of sea urchin and Dictyostelium dyneins. The N-terminal one-third of the heavy chain of rat brain (cytoplasmic) dynein shows high similarity (43.8% identical) to that of Dictyostelium cytoplasmic dynein but poor similarity (19.4% identical) to that of sea urchin flageliar dynein. These results suggested that the C-terminal two-thirds of the dynein molecule is conserved and plays an essential role in microtubule-dependent motility activity, whereas the N-terminal regions are different between cytoplasmic and flageilar dyneins.The nerve cell develops a polarized morphology composed of highly branched dendrites, a long axon, and synapses. Because of the lack of protein synthesis machinery in the axon, proteins in the axon and synapses must be transported down the axon after being synthesized in the cell body. Many proteins are transported bidirectionally in membranous organelles of various kinds by fast axonal flow (200-400 mm/ day), whereas other proteins such as cytoskeletal components are conveyed by slow axonal flow (0.5-2 mm/day). Electron microscopic studies of the neuronal cytoskeleton in vivo have suggested that microtubules and crossbridges between microtubules and membranous organelles form the structural basis for fast flow (1-3). Recently, two microtubule-activated ATPases, kinesin and brain dynein [cytoplasmic dynein, microtubule-associated protein (MAP) 1C], were identified as candidates for anterograde and retrograde molecular motors, respectively (4-7). In fact, kinesin is primarily associated with anterogradely moving membranous organelles in the axon, strongly supporting the hypothesis that kinesin is really an anterograde motor in vivo (8). Brain dynein is associated with both anterogradely and retrogradely moving membranous organelles (9), consistent with the suggestion that brain dynein is transported to the nerve terminal by fast flow and subsequently functions as a retrograde transport motor in vivo. Molecular genetic and ultrastructural approaches have dissected the molecular structure and functional domains of kinesin motors (10-12). However, detailed analysis of the primary structure of brain dynein hasThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. not yet been accomplished. To further elucidate the functi...

Section: Resultsmentioning

confidence: 85%

“…This region contains four putative ATP-binding motifs-i.e., P-loop consensus sequences of ATP-binding domain. (19,20). (B) Rat brain dynein heavy chain and Dictyostelium cytoplasmic dynein (18).…”

Section: Resultsmentioning

confidence: 99%

The primary structure of rat brain (cytoplasmic) dynein heavy chain, a cytoplasmic motor enzyme.

Zhang

Tanaka

Nonaka

et al. 1993

“…Among them, the heavy chain, which belongs to the AAAϩ (ATPases associated with diverse cellular activities) superfamily (8), is responsible for the motor activities of dynein (9,10). The heavy chain forms a ring-like head (11) composed of six tandemly linked AAAϩ modules (AAA1-AAA6) (8), among which the first four (AAA1-AAA4) contain nucleotidebinding/hydrolysis sites (12)(13)(14)(15). Multiple nucleotide-binding/ hydrolysis sites within a ring structure are a common feature of the AAAϩ superfamily proteins (see, for example, refs.…”

mentioning

confidence: 99%

The coordination of cyclic microtubule association/dissociation and tail swing of cytoplasmic dynein

Imamula

Kon

Ohkura

et al. 2007

140

The dynein motor domain is composed of a tail, head, and stalk and is thought to generate a force to microtubules by swinging the tail against the head during its ATPase cycle. For this ''power stroke,'' dynein has to coordinate the tail swing with microtubule association/dissociation at the tip of the stalk. Although a detailed picture of the former process is emerging, the latter process remains to be elucidated. By using the single-headed recombinant motor domain of Dictyostelium cytoplasmic dynein, we address the questions of how the interaction of the motor domain with a microtubule is modulated by ATPase steps, how the two mechanical cycles (the microtubule association/dissociation and tail swing) are coordinated, and which ATPase site among the multiple sites in the motor domain regulates the coordination. Based on steadystate and pre-steady-state measurements, we demonstrate that the two mechanical cycles proceed synchronously at most of the intermediate states in the ATPase cycle: the motor domain in the poststroke state binds strongly to the microtubule with a K d of Ϸ0.2 M, whereas most of the motor domains in the prestroke state bind weakly to the microtubule with a Kd of >10 M. However, our results suggest that the timings of the microtubule affinity change and tail swing are staggered at the recovery stroke step in which the tail swings from the poststroke to the prestroke position. The ATPase site in the AAA1 module of the motor domain was found to be responsible for the coordination of these two mechanical processes.AAAϩ protein ͉ ATPase cycle ͉ microtubule affinity ͉ motor protein ͉ power stroke

“…The C-terminal two-thirds contain six AAA ϩ modules (AAA1-AAA6) linked in tandem (9) and a seventh non-AAA ϩ module (14,15), all of which fold into a ring-shaped structure (AAA ring) (16) that is characteristic of AAA ϩ proteins (9). Each of the first four AAA ϩ modules (AAA1-AAA4) is predicted to contain a nucleotide binding͞hydrolysis site (17)(18)(19)(20), although ATPase activity at the AAA1 module is essential only for the motile activity of dynein (21), and others may regulate the ATPase cycle at AAA1 (21,22). The MT-binding site is in a small globular tip of a long (Ϸ15 nm) antiparallel coiled-coil protruding from the AAA ring (23,24).…”

mentioning

confidence: 99%

Two modes of microtubule sliding driven by cytoplasmic dynein

Shima

Kon

Imamula

et al. 2006

Dynein is a huge multisubunit microtubule (MT)-based motor, whose motor domain resides in the heavy chain. The heavy chain comprises a ring of six AAA (ATPases associated with diverse cellular activities) modules with two slender protruding domains, the tail and stalk. It has been proposed that during the ATP hydrolysis cycle, this tail domain swings against the AAA ring as a lever arm to generate the power stroke. However, there is currently no direct evidence to support the model that the tail swing is tightly linked to dynein motility. To address the question of whether the power stroke of the tail drives MT sliding, we devised an in vitro motility assay using genetically biotinylated cytoplasmic dyneins anchored on a glass surface in the desired orientation with a biotin-streptavidin linkage. Assays on the dyneins with the site-directed biotin tag at eight different locations provided evidence that robust MT sliding is driven by the power stroke of the tail. Furthermore, the assays revealed slow MT sliding independent of dynein orientation on the glass surface, which is mechanically distinct from the sliding driven by the power stroke of the tail.AAA protein ͉ motility ͉ motor protein ͉ power stroke