Controlling kinesin by reversible disulfide cross-linking. Identifying the motility-producing conformational change

Abstract

Conventional kinesin, a dimeric molecular motor, uses ATP-dependent conformational changes to move unidirectionally along a row of tubulin subunits on a microtubule. Two models have been advanced for the major structural change underlying kinesin motility: the first involves an unzippering/zippering of a small peptide (neck linker) from the motor catalytic core and the second proposes an unwinding/rewinding of the adjacent coiled-coil (neck coiled-coil). Here, we have tested these models using disulfide cross-linking of cysteines engineered into recombinant kinesin motors. When the neck linker motion was prevented by cross-linking, kinesin ceased unidirectional movement and only showed brief one-dimensional diffusion along microtubules. Motility fully recovered upon adding reducing agents to reverse the cross-link. When the neck linker motion was partially restrained, single kinesin motors showed biased diffusion towards the microtubule plus end but could not move effectively against a load imposed by an optical trap. Thus, partial movement of the neck linker suffices for directionality but not for normal processivity or force generation. In contrast, preventing neck coiled-coil unwinding by disulfide cross-linking had relatively little effect on motor activity, although the average run length of single kinesin molecules decreased by 30-50%. These studies indicate that conformational changes in the neck linker, not in the neck coiled-coil, drive processive movement by the kinesin motor.

Figure 2

Effects of neck linker cross-linking on kinesin motility. WT denotes K560 Cys-light kinesin without cysteine introduction. (a) Histograms of microtubule gliding velocities without and with DTNB treatment are shown. Arrowheads indicate mean value (Table ). (b) Histograms of run lengths of single molecular motility of neck linker cross-linked kinesins along axonemes visualized using total internal reflection fluorescent microscopy. The distributions were fitted with either an exponential curve or a Gaussian curve (see Materials and Methods). Arrowheads indicate the average run length (Table ). (c) Load-dependent velocity and dissociation rate of WT and Cys330/Cys4 molecules were determined by optical trapping beads assays. (Open circle) Without DTNB; (closed circle) with DTNB; (triangle) with DTNB at 10 times higher motor density (also marked with “M” to differentiate this multiple motor condition from single motor conditions [“S”]). Force–velocity curves shown here represent a lower bound for force production (see Materials and Methods).

Figure 1

Constructs for cross-linking of the neck region. (a) Three-dimensional structure of rat kinesin dimer (PDB No. 3KIN [Kozielski et al. 1997]; numbered as in human kinesin). The catalytic core (residues 2–322; gray), the neck linker (residues 323–336; red) and the neck coiled-coil (337–368; cyan) are shown. Positions of the mutations for cross-linking are shown by space filing of Cα atoms (Ser330Cys/Leu4Cys or Glu334Cys/Lys222Cys for cross-linking of the neck linker and Ala337Cys or Tyr344Cys for cross-linking of the neck coiled-coil). Inset shows a view from the reverse side. Smaller spheres indicate positions of Cβ atoms. (b) DTNB-induced disulfide cross-linking of neck linker mutants (Cys334/Cys222 and Cys330/Cys4), and (c) neck coiled-coil mutants (Cys344, Cys337, Cys337/Cys344) as analyzed by nonreducing SDS-PAGE. The DTNB concentration in c was 200 μM. Disulfide formation was reversed by subsequent treatment with 5 mM DTT (+DTT, right lane in c). The arrowheads show noncross-linked kinesin and the arrows show intramolecular cross-linked kinesin (b) and intermolecular cross-linked kinesin (c). The intramolecular cross-link for C334/C222 produces an increase in electrophoretic mobility, whereas the C330/C4 cross-link decreases mobility. No higher molecular weight band (i.e., intermolecular cross-linking) was detected for the neck linker mutants (not shown). The band indicated by open arrowhead (c) may represent a modified monomer or a dimer of degradation products.

Figure 1

Constructs for cross-linking of the neck region. (a) Three-dimensional structure of rat kinesin dimer (PDB No. 3KIN [Kozielski et al. 1997]; numbered as in human kinesin). The catalytic core (residues 2–322; gray), the neck linker (residues 323–336; red) and the neck coiled-coil (337–368; cyan) are shown. Positions of the mutations for cross-linking are shown by space filing of Cα atoms (Ser330Cys/Leu4Cys or Glu334Cys/Lys222Cys for cross-linking of the neck linker and Ala337Cys or Tyr344Cys for cross-linking of the neck coiled-coil). Inset shows a view from the reverse side. Smaller spheres indicate positions of Cβ atoms. (b) DTNB-induced disulfide cross-linking of neck linker mutants (Cys334/Cys222 and Cys330/Cys4), and (c) neck coiled-coil mutants (Cys344, Cys337, Cys337/Cys344) as analyzed by nonreducing SDS-PAGE. The DTNB concentration in c was 200 μM. Disulfide formation was reversed by subsequent treatment with 5 mM DTT (+DTT, right lane in c). The arrowheads show noncross-linked kinesin and the arrows show intramolecular cross-linked kinesin (b) and intermolecular cross-linked kinesin (c). The intramolecular cross-link for C334/C222 produces an increase in electrophoretic mobility, whereas the C330/C4 cross-link decreases mobility. No higher molecular weight band (i.e., intermolecular cross-linking) was detected for the neck linker mutants (not shown). The band indicated by open arrowhead (c) may represent a modified monomer or a dimer of degradation products.

Figure 5

Cross-linking of the neck coiled-coil using long cross-linkers. (a) Structures of the cross-linking agents oPDM and pPDM possessing two maleimide groups that react with thiols. (b) Cross-linking of neck coiled-coil mutants Cys344 and Cys337 using oPDM or pPDM analyzed by SDS-PAGE. Arrowhead and arrow show noncross-linked and cross-linked kinesin, respectively. The concentration of cross-linking agent was 100 μM. (c) Histogram of velocities of Cys337 after cross-linking, analyzed using the single molecule fluorescence motility assays. The arrows denote the mean velocities (19.1 ± 4.4, 19.4 ± 4.4, 19.3 ± 4.4 and 15.3 ± 4.9 μm/min for reduced, with DTNB-, oPDM-, and pPDM-treated, respectively). The shift in the distribution after cross-linking with pPDM most likely reflects the presence of both cross-linked and noncross-linked molecules.

Figure 3

Diffusional motion of neck linker-cross-linked kinesin molecules. (a) Typical displacement plots for cross-linked Cys334/Cys222 and Cys330/Cys4. Y axis indicates the displacement toward plus end of axonemes. The polarity was determined by the direction of continuous motion of occasional noncross-linked molecules in the preparation. The variation in the position of cross-linked Cys330/Cys4 with the nonhydrolyzable ATP analogue AMPPNP (1 mM) reflects the error in the tracking of single molecules and not actual motion. (b) MSD plots for Cys334/Cys222 with DTNB (red circle), Cys330/Cys4 without DTNB (square), with DTNB (blue circle), with DTNB in the presence of 1 mM AMPPNP (diamond), as shown with two different magnifications in y axis. Lines show theoretical curves. Cys334/Cys222+DTNB was fitted with MSD = 2Dt (D = 2.4 × 10⁻¹¹ cm²/s), and Cys330/Cys4 and Cys330/Cys4+DTNB were fitted with MSD = 2Dt + v ² t ² (D = 17 × 10⁻¹¹ and 2.6 × 10⁻¹¹ cm²/s and v = 21 and 3.2 μm/min, respectively; diffusion coefficient for Cys330/Cys4 without DTNB is overestimated due to the lack of temporal resolution). See supplemental videos 1 and 2, showing movements of Cys330/Cys4 without and with DTNB.

Figure 4

Effects of neck coiled-coil cross-linking on kinesin motility. (a) Histograms of microtubule gliding velocities without and with DTNB treatment are shown. Arrowheads indicate mean value (Table ). (b) Histograms of run lengths of single molecule motility of neck coiled-coil cross-linked kinesins along axonemes visualized using total internal reflection fluorescent microscopy. The run lengths were fit with an exponential curve. Arrowheads indicate average run length (Table ). (c) Load-dependent velocity and dissociation rate of Cys344 and Cys337/344 molecules were determined by optical trapping bead assays. (Open circle) Without DTNB; (closed circle) with DTNB. The increase in dissociation rate of Cys337/344 is statistically significant at lower forces, as tested by t test using dissociation rates obtained from 10 individual beads (P value of 0.018 and 0.015 at 0.75 and 1.25 pN, respectively). The increase in the dissociation rate of Cys344 was small (∼1.2-fold) and was not statistically significant at any load (P value of >0.3).

Figure 6

Schematic model showing the effects of neck linker cross-linking on kinesin movement. Two kinesin's catalytic cores are shown in blue and green, and the adjacent neck linker and the neck coiled-coil are shown in black lines. α- and β-tubulin subunits are shown in gray with the flexible COOH terminus region shown by an orange line. T and D denote ATP and ADP, respectively. (a) Model for processive movement of kinesin as described previously (Rice et al. 1999; Vale and Milligan 2000). Upon binding of ATP to the microtubule-bound head, the neck linker docks onto the catalytic core and moves partner head toward plus end. The forward head searches and binds to a new tubulin-binding site to achieve two-head–bound intermediate. Kinesin can repeat this cycle more than 100 times before detachment from the microtubule. (b) Cross-linking of the neck linker at the middle (Cys330/Cys4) enables directional motion due to a small power stroke but not processive motion. Upon binding of ATP, a part of the neck linker in the microtubule-bound head folds (red arrows). However, the access of the partner head to a tubulin-binding site is inhibited until the bound head detaches. The motor partitions between a weak binding state in which it can diffuse along the microtubule and a strong binding state in which it can undergo a short stroke. (c) Disulfide cross-linking of the neck linker at the tip of the catalytic core (Cys334/Cys222) abolishes motile activity, since the neck linker cannot change position. Weak electrostatic interaction between the COOH terminus of tubulin and the initial portion of the neck coiled-coil allows the molecule to diffuse along the microtubule (see also Thorn et al. 2000).