Key residues on microtubule responsible for activation of kinesin ATPase

Abstract

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 alpha-tubulin, and the negatively charged residues in H12 of beta-tubulin. Mutation in the alpha-tubulin-binding site results in a deceleration of ATP hydrolysis (k(cat)), whereas mutation in the beta-tubulin-binding site lowers the affinity for MTs (K(0.5)MT). The residue E415 in alpha-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 alpha-E415 could transmit a signal to the kinesin nucleotide pocket, triggering its conformational change and leading to the release of ADP.

Figure 1

Design of mutants and their phenotypes. (A) The positions of the charged residues in the area of the MT spanning H11 and H12, in both α- and β-tubulin, targeted for alanine mutagenesis. Each of the positively (red) or negatively charged (blue) residues was substituted with alanine, which gave rise to 36 mutant strains in total. The resultant phenotypes of yeast mutant cells are marked by the letter L or S, indicating haploid lethal or slow growth, respectively. (B) A ribbon diagram of a tubulin dimer viewed from the putative outside of the MT with its minus end to the right (Lowe et al, 2001). The positions of the charged residues are marked by the same coloring scheme as in (A).

Figure 2

Binding frequency, run length, and velocity of HK560-Cy3 measured with mutant MTs. The binding frequency, mean run length, and velocity were calculated as described in Materials and methods section. NA stands for data not available and the error bars represent s.e.m. values. The mutants β-E410A, D417A, and E421A were previously characterized in different assay condition (Uchimura et al, 2006). The distributions of the raw data are available in Supplementary Figure S2.

Figure 3

Unbinding force and stall force of kinesin measured using mutant MTs. (A–C) Unbinding force of single-headed kinesin was measured with load applied toward the MT minus end (A) in the presence of ADP, (B) in the absence of nucleotides, and (C) in the presence of AMP-PNP. The asterisks mark the mutants in critical residues. The distributions of the unbinding forces for each mutant for both minus- and plus-end loadings are shown in Supplementary Figure S3A–C. The stiffness of the optical trap was 0.076 pN nm⁻¹. The error bars represent s.e.m. values. (D) Kinesin stall force for conventional, two-headed kinesin was measured using optical tweezers at trap stiffness of 0.076 pN nm⁻¹. Each bar represents the average stall force with s.e.m. value. The distributions of stall force are shown in Supplementary Figure S3D. (E) The stall force plotted against the unbinding force for minus-end loading measured in three nucleotide conditions. The stall force plotted against the unbinding force for plus-end loading is available in Supplementary Figure S4. Note that the absolute amount of unbinding force depends on the loading rate and, therefore, cannot be equal to the stall force (Kawaguchi et al, 2003). The mutants β-E410A, D417A, and E421A were previously characterized in different assay condition.

Figure 4

Steady-state ATPase activity of kinesin (HK560) with mutant MTs. The data shown in this graph are a representative example of the measurements for wild-type and mutant MTs. The maximal turnover rate (k_cat) and apparent Michaelis–Menten constant (K_0.5MT) were calculated by fitting the data to a hyperbola, and the averages obtained from four to six sets of experiments are shown in Table I.

Figure 5

Mictrotubule dependence of mant-ADP release from HK560 kinesin. To determine the ADP release rate, mant-ADP-loaded HK560 was mixed with MTs in a stopped-flow apparatus at post-mixing concentrations of 1 μM HK560, 2 μM mant-ADP, 0.25–10 μM wild-type or α-E415A MTs, and 1 mM ATP. The apparent rate of ADP dissociation was derived by fitting the fluorescence decay by double exponentials, giving fast- and slow-phase rates for ADP release (see Materials and methods section for details). Fast-phase rates (k_obs) are plotted as a function of MT concentration. Slow-phase rates were low and independent of MT concentrations, and are omitted from the graphic and analysis. MT dependence of k_obs was fit to a hyperbolic model, from which values for parameters k_max and K_0.5MT were extracted (Table II).

Figure 6

Structural basis for MT-dependent activation of kinesin ATPase. (A) Two independent sites on the MT critical for kinesin motility: the negatively charged residues spanning the H11–12 loop and H12 of α-tubulin (highlighted in red and purple), and the negatively charged residues in H12 of β-tubulin (orange). A tubulin dimer is viewed from the putative outside of the MT tube with its minus end to the right. (B–D) Schematic model for kinesin interaction with MTs in different nucleotide states. In the diffusional binding state (B), kinesin, tightly holding ADP, is in search of the next binding site through diffusion along the MT. Nonspecific electrostatic interaction between the negatively charged C-terminal tail of tubulin (CTT) and the positively charged interface of kinesin contributes to this weak interaction. When the switch II helix/loop of kinesin (red) encounters the H11–12 loop/H12 of α-tubulin (C), kinesin changes its conformation and ejects ADP from the nucleotide pocket. After, or concomitant with ADP release (D), kinesin is further locked on the MT through the interaction between L8/α5 of kinesin (orange) and H12 of β-tubulin. This stable interaction enables kinesin to sustain load.