Tomohiro Shima

The 2.8 A crystal structure of the dynein motor domain

Dyneins are microtubule-based AAA+ motor complexes that power ciliary beating, cell division, cell migration and intracellular transport. Here we report the most complete structure obtained so far, to our knowledge, of the 380-kDa motor domain of Dictyostelium discoideum cytoplasmic dynein at 2.8 Å resolution; the data are reliable enough to discuss the structure and mechanism at the level of individual amino acid residues. Features that can be clearly visualized at this resolution include the coordination of ADP in each of four distinct nucleotide-binding sites in the ring-shaped AAA+ ATPase unit, a newly identified interaction interface between the ring and mechanical linker, and junctional structures between the ring and microtubule-binding stalk, all of which should be critical for the mechanism of dynein motility. We also identify a long-range allosteric communication pathway between the primary ATPase and the microtubule-binding sites. Our work provides a framework for understanding the mechanism of dynein-based motility.

Two modes of microtubule sliding driven by cytoplasmic dynein.

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.

Biomolecular-Motor-Based Nano- or Microscale Particle Translocations on DNA Microarrays

We aimed to create autonomous on-chip systems that perform targeted translocations of nano- or microscale particles in parallel using machinery that mimics biological systems. By exploiting biomolecular-motor-based motility and DNA hybridization, we demonstrate that single-stranded DNA-labeled microtubules gliding on kinesin-coated surfaces acted as cargo translocators and that single-stranded DNA-labeled cargoes were loaded/unloaded onto/from gliding microtubules at micropatterned loading/unloading sites specified by DNA base sequences. Our results will help to create autonomous molecular sorters and sensors.

Molecular mechanism of force generation by dynein, a molecular motor belonging to the AAA+ family

Dynein is an AAA+ (ATPase associated with various cellular activities)-type motor complex that utilizes ATP hydrolysis to actively drive microtubule sliding. The dynein heavy chain (molecular mass >500 kDa) contains six tandemly linked AAA+ modules and exhibits full motor activities. Detailed molecular dissection of this motor with unique architecture was hampered by the lack of an expression system for the recombinant heavy chain, as a result of its large size. However, the recent success of recombinant protein expression with full motor activities has provided a method for advances in structure-function studies in order to elucidate the molecular mechanism of force generation.

C-sequence of the Dictyostelium cytoplasmic dynein participates in processivity modulation

We examined the functional roles of C‐sequence, a 47‐kDa non‐AAA+ module at the C‐terminal end of the 380‐kDa Dictyostelium dynein motor domain. When the distal segment of the C‐sequence was deleted from the motor domain, the single‐molecule processivity of the dimerized motor domain was selectively impaired without its ensemble motile ability and ATPase activity being severely affected. When the hinge‐like sequence between the distal and proximal C‐sequence segments was made more or less flexible, the dimeric motor showed lower or higher processivity, respectively. These results suggest a potential function of the distal C‐sequence segment as a modulator of processivity.

Direct observation shows superposition and large scale flexibility within cytoplasmic dynein motors moving along microtubules

Cytoplasmic dynein is a dimeric AAA+ motor protein that performs critical roles in eukaryotic cells by moving along microtubules using ATP. Here using cryo-electron microscopy we directly observe the structure of Dictyostelium discoideum dynein dimers on microtubules at near-physiological ATP concentrations. They display remarkable flexibility at a hinge close to the microtubule binding domain (the stalkhead) producing a wide range of head positions. About half the molecules have the two heads separated from one another, with both leading and trailing motors attached to the microtubule. The other half have the two heads and stalks closely superposed in a front-to-back arrangement of the AAA+ rings, suggesting specific contact between the heads. All stalks point towards the microtubule minus end. Mean stalk angles depend on the separation between their stalkheads, which allows estimation of inter-head tension. These findings provide a structural framework for understanding dyneins directionality and unusual stepping behaviour.

Protein Engineering Approaches to Study the Dynein Mechanism using a Dictyostelium Expression System

Dyneins are microtubule-based motor complexes that power a wide variety of motile processes within eukaryotic cells, including the beating of cilia and flagella and intracellular trafficking along microtubules. Mechanistic studies on dynein have been hampered by their enormous size (molecular masses of 0.5-3MDa) and molecular complexity. However, the recent establishment of recombinant expression systems for cytoplasmic dynein, together with structural and functional analyses, has advanced our understanding of the molecular mechanisms of dynein motility. Here, we describe several protocols for protein engineering approaches to the dynein mechanism using a Dictyostelium discoideum expression system. We first describe the design and preparation of recombinant dynein suitable for mechanistic studies. We then discuss two distinct functional assays that take advantage of the recombinant dynein. One is for detection of dyneins conformational changes during the ATPase cycle. Another is an in vitro motility assay at multiple- and single-molecule levels for examination of the dynamic behavior of dynein moving on a microtubule.

Biomolecular motor-based cargo transporters with loading/unloading mechanisms on a micro-patterned DNA array

This paper describes research in creating autonomous cargo transporters that load, transport, and unload cargo molecules using the machinery in living cells. By exploiting biomolecular motor-based motility and DNA hybridization, we constructed autonomous cargo transporters and demonstrated that kinesin-driven microtubules can selectively load and transport cargoes toward micro- patterned DNA spots designed to be unloading sites and selectively unload the transported cargoes at the DNA spots specified by particular DNA base sequences. These autonomous operations may help create highly miniaturized on-chip-systems such as molecular sorters and sensors, and may also provide an alternative technology to pressure-driven or electrokinetic flow-based microfluidic devices.

An autonomous molecular transport system using DNAs and motor proteins in molecular communication

This paper describes a molecular transport system in molecular communication that uses the machinery in living cells. The molecular transport system requires: 1) loading of the specified cargo molecules at a loading site (at a sender); 2) transport of the loaded cargoes to an unloading site (to a receiver); and 3) unloading of the transported cargoes at the unloading site, all without using external stimuli. Through the DNA strand exchange at a loading site and at an unloading site, and through motility of a biological motor system (kinesins and microtubules), the authors of this paper constructed a molecular transport system and demonstrated that kinesin-driven microtubules autonomously load, transport and unload cargoes to which a specified DNA strand is attached.

Real-time observation of flexible domain movements in Cas9

The CRISPR-associated protein Cas9 is a widely used genome editing tool that recognizes and cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have demonstrated the multi-domain architecture of Cas9 and sequential domain movements upon binding to sgRNA and the target DNA. These studies also have hinted at flexibility between the domains, but whether these flexible movements take place under dynamic and physiological conditions is unclear. Here, we directly observed dynamic fluctuations by multiple Cas9 domains using single-molecule FRET. The flexible domain movements allow Cas9 to take transient conformations beyond those revealed by crystal structures. Importantly, one Cas9 nuclease domain accessed to the DNA cleavage position only during such flexible movement, suggesting the importance of this flexibility in the DNA cleavage process. Moreover, changes in domain flexibility in the presence of nucleic acids indicated that flexible Cas9 domain movements are involved in the sgRNA and the target DNA binding processes. Collectively, our results highlight the potential role of fluctuations in driving Cas9 catalytic processes.