Yosuke Nishikawa

Structure of the entire stalk region of the Dynein motor domain.

Dyneins are large microtubule-based motor complexes that power a range of cellular processes including the transport of organelles, as well as the beating of cilia and flagella. The motor domain is located within the dynein heavy chain and comprises an N-terminal mechanical linker element, a central ring of six AAA+ modules of which four bind or hydrolyze ATP, and a long stalk extending from the AAA+ring with a microtubule-binding domain (MTBD) at its tip. A crucial mechanism underlying the motile activity of cytoskeletal motor proteins is precise coupling between the ATPase and track-binding activities. In dynein, a stalk region consisting of a long (~15nm) antiparallel coiled coil separates these two activities, which must facilitate communication between them. This communication is mediated by a small degree of helix sliding in the coiled coil. However, no high-resolution structure is available of the entire stalk region including the MTBD. Here, we have reported the structure of the entire stalk region of mouse cytoplasmic dynein in a weak microtubule-binding state, which was determined using X-ray crystallography, and have compared it with the dynein motor domain from Dictyostelium discoideum in a strong microtubule-binding state and with a mouse MTBD with its distal portion of the coiled coil fused to seryl-tRNA synthetase from Thermus thermophilus. Our results strongly support the helix-sliding model based on the complete structure of the dynein stalk with a different form of coiled-coil packing. We also propose a plausible mechanism of helix sliding together with further analysis using molecular dynamics simulations. Our results present the importance of conserved proline residues for an elastic motion of stalk coiled coil and imply the manner of change between high-affinity state and low-affinity state of MTBD.

A flipped ion pair at the dynein-microtubule interface is critical for dynein motility and ATPase activation

Salt bridges at the dynein–microtubule interface couple microtubule binding to ATPase activation and thereby control the directional movement of dynein

Structural Change in the Dynein Stalk Region Associated with Two Different Affinities for the Microtubule

Dynein is a large microtubule-based motor complex that requires tight coupling of intra-molecular ATP hydrolysis with the generation of mechanical force and track-binding activity. However, the microtubule-binding domain is structurally separated by about 15nm from the nucleotide-binding sites by a coiled-coil stalk. Thus, long-range two-way communication is necessary for coordination between the catalytic cycle of ATP hydrolysis and dyneins track-binding affinities. To investigate the structural changes that occur in the dynein stalk region to produce two different microtubule affinities, here we improve the resolution limit of the previously reported structure of the entire stalk region and we investigate structural changes in the dynein stalk and strut/buttress regions by comparing currently available X-ray structures. In the light of recent crystal structures, the basis of the transition from the low-affinity to the high-affinity coiled-coil registry is discussed. A concerted movement model previously reported by Carter and Vale is modified more specifically, and we proposed it as the open zipper model.

A Well-Defined Osmium-Cupin Complex: Hyperstable Artificial Osmium Peroxygenase

Thermally stable TM1459 cupin superfamily protein from Thermotoga maritima was repurposed as an osmium (Os) peroxygenase by metal-substitution strategy employing the metal-binding promiscuity. This novel artificial metalloenzyme bears a datively bound Os ion supported by the 4-histidine motif. The well-defined Os center is responsible for not only the catalytic activity but also the thermodynamic stability of the protein folding, leading to the robust biocatalyst (Tm ≈ 120 °C). The spectroscopic analysis and atomic resolution X-ray crystal structures of Os-bound TM1459 revealed two types of donor sets to Os center with octahedral coordination geometry. One includes trans-dioxide, OH, and mer-three histidine imidazoles (O3N3 donor set), whereas another one has four histidine imidazoles plus OH and water molecule in a cis position (O2N4 donor set). The Os-bound TM1459 having the latter donor set (O2N4 donor set) was evaluated as a peroxygenase, which was able to catalyze cis-dihydroxylation of several alkenes efficiently. With the low catalyst loading (0.01% mol), up to 9100 turnover number was achieved for the dihydroxylation of 2-methoxy-6-vinyl-naphthalene (50 mM) using an equivalent of H2O2 as oxidant at 70 °C for 12 h. When octene isomers were dihydroxylated in a preparative scale for 5 h (2% mol cat.), the terminal alkene octene isomers was converted to the corresponding diols in a higher yield as compared with the internal alkenes. The result indicates that the protein scaffold can control the regioselectivity by the steric hindrance. This protein scaffold enhances the efficiency of the reaction by suppressing disproportionation of H2O2 on Os reaction center. Moreover, upon a simple site-directed mutagenesis, the catalytic activity was enhanced by about 3-fold, indicating that Os-TM1459 is evolvable nascent osmium peroxygenase.