Yusuke Oguchi

Load-dependent ADP binding to myosins V and VI: Implications for subunit coordination and function

Dimeric myosins V and VI travel long distances in opposite directions along actin filaments in cells, taking multiple steps in a “hand-over-hand” fashion. The catalytic cycles of both myosins are limited by ADP dissociation, which is considered a key step in the walking mechanism of these motors. Here, we demonstrate that external loads applied to individual actomyosin V or VI bonds asymmetrically affect ADP affinity, such that ADP binds weaker under loads assisting motility. Model-based analysis reveals that forward and backward loads modulate the kinetics of ADP binding to both myosins, although the effect is less pronounced for myosin VI. ADP dissociation is modestly accelerated by forward loads and inhibited by backward loads. Loads applied in either direction slow ADP binding to myosin V but accelerate binding to myosin VI. We calculate that the intramolecular load generated during processive stepping is ≈2 pN for both myosin V and myosin VI. The distinct load dependence of ADP binding allows these motors to perform different cellular functions.

Identification of a strong binding site for kinesin on the microtubule using mutant analysis of tubulin.

The kinesin‐binding site on the microtubule has not been identified because of the technical difficulties involved in the mutant analyses of tubulin. Exploiting the budding yeast expression system, we succeeded in replacing the negatively charged residues in the α‐helix 12 of β‐tubulin with alanine and analyzed their effect on kinesin‐microtubule interaction in vitro. The microtubule gliding assay showed that the affinity of the microtubules for kinesin was significantly reduced in E410A, D417A, and E421A, but not in E412A mutant. The unbinding force measurement revealed that in the former three mutants, the kinesin‐microtubule interaction in the adenosine 5′‐[β,γ‐imido]triphosphate state (AMP‐PNP state) became less stable when a load was imposed towards the microtubule minus end. In parallel with this decreased stability, the stall force of kinesin was reduced. Our results implicate residues E410, D417, and E421 as crucial for the kinesin‐microtubule interaction in the strong binding state, thereby governing the size of kinesin stall force.

Key residues on microtubule responsible for activation of kinesin ATPase

Microtubule (MT) binding accelerates the rate of ATP hydrolysis in kinesin. To understand the underlying mechanism, using charged‐to‐alanine mutational analysis, we identified two independent sites in tubulin, which are critical for kinesin motility, namely, a cluster of negatively charged residues spanning the helix 11–12 (H11–12) loop and H12 of α‐tubulin, and the negatively charged residues in H12 of β‐tubulin. Mutation in the α‐tubulin‐binding site results in a deceleration of ATP hydrolysis (kcat), whereas mutation in the β‐tubulin‐binding site lowers the affinity for MTs (K0.5MT). The residue E415 in α‐tubulin seems to be important for coupling MT binding and ATPase activation, because the mutation at this site results in a drastic reduction in the overall rate of ATP hydrolysis, largely due to a deceleration in the reaction of ADP release. Our results suggest that kinesin binding at a region containing α‐E415 could transmit a signal to the kinesin nucleotide pocket, triggering its conformational change and leading to the release of ADP.

The role of tropomyosin domains in cooperative activation of the actin-myosin interaction

To establish α-tropomyosin (Tm)s structure-function relationships in cooperative regulation of muscle contraction, thin filaments were reconstituted with a variety of Tm mutants (Δ2Tm, Δ3Tm, Δ6Tm, P2sTm, P3sTm, P2P3sTm, P1P5Tm, and wtTm), and force and sliding velocity of the thin filament were studied using an in vitro motility assay. In the case of deletion mutants, Δ indicates which of the quasi-equivalent repeats in Tm was deleted. In the case of period (P) mutants, an Ala cluster was introduced into the indicated period to strengthen the Tm-actin interaction. In P1P5Tm, the N-terminal half of period 5 was substituted with that of period 1 to test the quasi-equivalence of these two Tm periods. The reconstitution included bovine cardiac troponin. Deletion studies revealed that period 3 is important for the positive cooperative effect of Tm on actin filament regulation and that period 2 also contributes to this effect at low ionic strength, but to a lesser degree. Furthermore, Tm with one extra Ala cluster at period 2 (P2s) or period 3 (P3s) did not increase force or velocity, whereas Tm with two extra Ala clusters (P2P3s) increased both force and velocity, demonstrating interaction between these periods. Most mutants did not move in the absence of Ca(2+). Notable exceptions were Δ6Tm and P1P5Tm, which moved near at the full velocity, but with reduced force, which indicate impaired relaxation. These results are consistent with the mechanism that the Tm-actin interaction cooperatively affects actin to result in generation of greater force and velocity.

Mass spectrometric screening of ligands with lower off-rate from a clicked-based pooled library.

This paper describes a convenient screening method using ion trap electrospray ionization mass spectrometry to classify ligands to a target molecule in terms of kinetic parameters. We demonstrate this method in the screening of ligands to a hexahistidine tag from a pooled library synthesized by click chemistry. The ion trap mass spectrometry analysis revealed that higher stabilities of ligand-target complexes in the gas phase were related to lower dissociation rate constants, i.e., off-rates in solution. Finally, we prepared a fluorescent probe utilizing the ligand with lowest off-rate and succeeded in performing single molecule observations of hexahistidine-tagged myosin V walking on actin filaments.

The Force Production by the Depolymerization Activity of MCAK

During cell division the replicating chromosomes must be precisely arranged and separated polewards. Though many cellular processes involving motility require force generation by motor proteins, the chromosome movement is suggested to use the energy stored at plus ends of the microtubules to which kinetochores are attached. This energy is converted into the chromosome movement via passive couplers, whereas the role of the microtubule-based motors is supposed to be limited to the regulation of microtubule dynamics. The microtubule-depolymerizing kinesins, such as mitotic centromere-associated kinesin (MCAK), which is a founding member of kinesin-13 family, facilitate microtubule dynamics in a spindle and are required for the chromosome congression and segregation; however, the key question - whether the depolymerizing activity generates tension to pull the chromosomes - has remained unsolved. To probe the link between the generation of tension and the microtubule-disassembling activity of MCAK, we developed a single-molecule assay to examine the interaction between a single microtubule and multiple MCAK molecules under the fluorescence microscope equipped with the dual-trap optical tweezers. Here we show that the microtubule-disassembling activity of MCAK generates significant tension. The depolymerization force increases with the number of interacting MCAK molecules. These results provide a simple and attractive model for the generation of the driving force and the regulation of chromosome segregation in a spindle by the activity of MCAK.