(1) A flipped ion pair at the dynein-microtubule interface is critical for dynein motility and ATPase activation
We have made important steps toward understanding how dynein—a "molecular motor"—walks along tube-like structure in the cell to move cellular cargo from the outer structures toward the cell body of neurons. The action of this molecule is important for a number of cell functions including axonal transport and chromosome segregation, and its dysfunction is known to lead to a congenital developmental brain disorder known as lissencephaly.
Though cells may look like shapeless blobs of liquid encased in a membrane, in fact they have a complex skeleton-like structure, known as the cytoskeleton, made up of filaments called microtubules. Motor proteins, which include dynein and kinesin, can move along these tubules to transport cargo into and out of the center of the cell. The motor proteins use an energy-currency molecule, ATP, to power their movements along the microtubules. The motor proteins hydrolyze ATP to ADP, and convert the released chemical energy to mechanical energy which is used for movement. The mechanism is quite well understood for kinesin, but in the case of dynein, it has been difficult to explain how communication takes place between the site of microtubule binding and the site of ATP hydrolysis, which are relatively far from each other, separated by a stalk.
Schematic representation of the dynein-microtubule complex showing he structural elements likely to be involved in allosteric communication between the microtubule and the ATPase site in dynein
Graph showing the movement over time of wild type and mutant types along the microtubules in the presence of 1 mM ATP. The mutants can be seen to be going back and forth.
We used cryo electron microscopy—where molecules are cooled to very low temperatures in the microscope—and examined the structure of dynein on the microtubule. The result revealed that two specific amino acid residues on the microtubule structure, R403 and E416, are key to turning on the switch that is critical for the activation of the dynein motor—demonstrating that when mutations in these sequences are present, the dynein fails to achieve directional movement on the microtubule, ending up simply moving back and forth in a random fashion. This lends weight to the idea, that has been generally accepted, that the motion of molecular motors is basically driven by random, Brownian motion, and that motors are able to move in one direction thanks to subtle changes in the strength of bonds at the motor-microtubule interface.
Additionally, we discovered that turning on the mechanical switch at the motor-microtubule interface leads to ATP hydrolysis. These results altogether indicate that the subtle structural changes in the bonds at the interface are transmitted through a small change in the structure of the stalk—there are two coils that link the two binding regions, and a small shift in the configuration of the coils gives the cue for ATP hydrolysis at the ATP binding site.
The figures on the left and right show a model of the microtubule binding domain-microtubule interaction in the weak and strong binding states, respectively.
In the weak binding state, the microtubule binding domain exhibited diffusional motion constrained in the electronegative potential valley of the microtubule, with its electropositive surface orienting toward it. In the strong binding state (right), navigated by the flipped ion pair of E3390 and R403, the microtubule binding domain found its binding site on the microtubule. The molecular structures of the microtubule binding sites and the alpha-and beta-tubulin (which make up the microtubule) at the interface in the respective states are shown in the insets.
We were able to clearly demonstrate that the dynein molecular motor is activated by a 'switch' that controls mutual interactions between dynein and the microtubule. This is important, as a mutation in the structure of the switch has been demonstrated to cause lissencephaly, a congenital disorder. In the future, we hope that further understanding the interplay between dynein and microtubule, as this could pave the way for therapies for these conditions.
A flipped ion pair at the dynein-microtubule interface is critical for dynein motility and ATPase activation.
Seiichi Uchimura, Takashi Fujii, Hiroko Takazaki, Rie Ayukawa, Yosuke Nishikawa, Itsushi Minoura, You Hachikubo, Genji Kurisu, Kazuo Sutoh, Takahide Kon, Keiichi Namba, Etsuko Muto.
(2) Key residues on microtubule responsible for activation of kinesin ATPase
The ATPase activity of kinesin is enhanced by the binding of microtubules. By mutational analysis of tubulin using the budding yeast, Saccharomyces cerevisiae, we aimed to understand the structural basis how the microtubule binding leads to the activation of kinesin ATPase.
The charged residues contained in the area of microtubule spanning H11, the H11–12 loop, and H12 in both α- and β-tubulin were selected for mutagenesis to alanine. When the mutated tubulins were expressed in the yeast cells (a total of 36 mutant strains), some of the mutant strains became haploid lethal or resulted in the slow growth. We isolated the mutated microtubules from these strains and examined their interaction with kinesin in single-molecule motility assay. The result revealed six amino acids critical for kinesin motility (Fig. A).
Furthermore, the measurement of microtubule-activated kinesin ATPase showed a drastic reduction in E415A mutant in α-tubulin (Fig. B), which largely owes to a deceleration in the reaction of ADP release (Fig. C).
Our results suggest that the residue E415 of α-tubulin is important for the coupling of microtubule binding and ATPase activation. The interaction through E415 of α-tubulin could transmit a signal to the kinesin nucleotide pocket, triggering its conformational change and leading to the release of ADP (Fig. D). In future studies, we hope to clarify the structural pathway of communication in kinesin connecting the microtubule binding site and the nucleotide pocket, and eventually understand the mechanism of mechano-chemical coupling.
