Rapid double 8-nm steps by a kinesin mutant

The mechanism by which conventional kinesin walks along microtubules is poorly understood, but may involve alternate binding to the microtubule and hydrolysis of ATP by the two heads. Here we report a single amino-acid change that affects stepping by the motor. Under low force or low ATP concentration, the motor moves by successive 8-nm steps in single-motor laser-trap assays, indicating that the mutation does not alter the basic mechanism of kinesin walking. Remarkably, under high force, the mutant motor takes successive 16-nm displacements that can be resolved into rapid double 8-nm steps with a short dwell between steps, followed by a longer dwell. The alternating short and long dwells under high force demonstrate that the motor stepping mechanism is inherently asymmetric, revealing an asymmetric phase in the kinesin walking cycle. Our findings support an asymmetric two-headed walking model for kinesin, with cooperative interactions between the two heads. The sensitivity of the 16-nm displacements to nucleotide and load raises the possibility that ADP release is a force-producing event of the kinesin cycle.     

Motility of single one-headed kinesin molecules along microtubules

The motility of single one-headed kinesin molecules (K351 and K340), which were truncated fragments of Drosophila two-headed kinesin, has been tested using total internal reflection fluorescence microscopy. One-headed kinesin fragments moved continuously along the microtubules. The maximum distance traveled until the fragments dissociated from the microtubules for both K351 and K340 was approximately 600 nm. This value is considerably larger than the space resolution of the measurement system (SD approximately 30 nm). Although the movements of the fragments fluctuated in forward and backward directions, statistical analysis showed that the average movements for both K340 and K351 were toward the plus end of the microtubules, i.e., forward direction. When BDTC (a 1.3-S subunit of Propionibacterium shermanii transcarboxylase, which binds weakly to a microtubule), was fused to the tail (C-terminus) of K351, its movement was enhanced, smooth, and unidirectional, similar to that of the two-headed kinesin fragment, K411. However, the travel distance and velocity of K351BDTC molecules were approximately 3-fold smaller than that of K411. These observations suggest that a single kinesin head has basal motility, but coordination between the two heads is necessary for stabilizing the basal motility for the normal level of kinesin processivity.     

Thermal activation of single kinesin molecules with temperature pulse microscopy

Conventional kinesin is a processive motor protein that keeps "walking" along a microtubule using chemical energy released by ATP hydrolysis. We previously studied the effects of temperature between 15 degrees and 35 degrees C on the moving velocity, force, and processivity of single kinesin molecules using a bead assay [Kawaguchi and Ishiwata, 2000b: Biochem Biophys Res Commun 272:895-899]. However, we could not examine the effects of temperature higher than 35 degrees C because of the thermal damage to proteins. Here, using temperature pulse microscopy (TPM) [Kato et al., 1999: Proc Natl Acad Sci USA 96:9602-9606], we could examine the temperature dependence of the gliding velocity of single kinesin molecules interacting with a microtubule above 35 degrees C up to 50 degrees C (instantaneously, approximately 60 degrees C), where the velocity reached 3.68 microm/s, the highest ever reported. The Arrhenius plot showed no breaks between 15 degrees and 50 degrees C with a unique activation energy of about 50 kJ/mol, suggesting that the molecular mechanism of kinesin motility is common over a broad temperature range including physiological temperature.     

Chemomechanical coupling of the forward and backward steps of single kinesin molecules

The molecular motor kinesin travels processively along a microtubule in a stepwise manner. Here we have studied the chemomechanical coupling of the hydrolysis of ATP to the mechanical work of kinesin by analysing the individual stepwise movements according to the directionality of the movements. Kinesin molecules move primarily in the forward direction and only occasionally in the backward direction. The hydrolysis of a single ATP molecule is coupled to either the forward or the backward movement. This bidirectional movement is well described by a model of Brownian motion assuming an asymmetric potential of activation energy. Thus, the stepwise movement along the microtubule is most probably due to Brownian motion that is biased towards the forward direction by chemical energy stored in ATP molecules.     

Mechanics of single kinesin molecules measured by optical trapping nanometry.

We have analyzed the mechanics of individual kinesin molecules by optical trapping nanometry. A kinesin molecule was adsorbed onto a latex bead, which was captured by an optical trap and brought into contact with an axoneme that was bound to a glass surface. The displacement of kinesin during force generation was determined by measuring the position of the beads with nanometer accuracy. As the displacement of kinesin was attenuated because of the compliance of the kinesin-to-bead and kinesin-to-microtubule linkages, the compliance was monitored during force generation and was used to correct the displacement of kinesin. Thus the velocity and the unitary steps could be obtained accurately over a wide force range. The force-velocity curves were linear from 0 to a maximum force at 10 microM and 1 mM ATP, and the maximum force was approximately 7 pN, which is larger by approximately 30% than values previously reported. Kinesin exhibited forward and occasionally backward stepwise displacements with a size of approximately 8 nm. The histograms of step dwell time show a monotonic decrease with time. Model calculations indicate that each kinesin head steps by 16-nm, whereas kinesin molecule steps by 8-nm.     

Dissecting the kinematics of the kinesin step

Kinesin walks processively on microtubules in an asymmetric hand-over-hand manner with each step spanning 16 nm. We used molecular simulations to determine the fraction of a single step due to conformational changes in the neck linker, and that due to diffusion of the tethered head. Stepping is determined largely by two energy scales, one favoring neck-linker docking and the other, ε(h)(MT-TH), between the trailing head (TH) and the microtubule. Neck-linker docking and an optimal value of ε(h)(MT-TH) are needed to minimize the probability that the TH takes side steps. There are three major stages in the kinematics of a step. In the first, the neck linker docks, resulting in ～(5-6) nm movements of the trailing head. The TH moves an additional (6-8) nm in stage II by anisotropic translational diffusion. In the third stage, spanning ～(3-4) nm, the step is complete with the TH binding to the αβ-tubulin binding site.     

Biased binding of single molecules and continuous movement of multiple molecules of truncated single-headed kinesin

Conventional kinesin has a double-headed structure consisting of two motor domains and moves processively along a microtubule using the two heads cooperatively. The movement of single and multiple truncated heads of Drosophila kinesin was measured using a laser trap and nanometer detecting apparatus. Single molecules of single-headed kinesin bound to the microtubules with a 3.5 nm biased displacement toward the plus end of the microtubule. The position of these single-headed kinesin molecules bound to a microtubule did not change until they had dissociated, indicating that single kinesin heads utilize nonprocessive movement processes. Two molecules of single-headed kinesin moved continuously along a microtubule with a lower velocity and force than that of single molecules of double-headed kinesin. The biased binding of the heads determines the directionality of movement, whereas two molecules of single-headed kinesin move continuously without dissociation from a microtubule.     

A kinetic study of the kinesin ATPase.

The mechanism of kinesin ATPase has been investigated by transient state kinetic analysis. The results satisfy the scheme [formula: see text] where T, D, and P(i) refer to nucleotide tri- and diphosphate and inorganic phosphate, respectively. The nucleotide-binding steps were measured by the fluorescence enhancement of mant (2'-(3')-O-(N-methylanthraniloyl)-ATP and mant-ADP. The initial rapid equilibrium binding steps (1) and (6) are followed by isomerizations (k2 = 170 +/- 30 s-1 at 20 degrees C, k-5 greater than 100 s-1). The increase in fluorescence is 20-25% larger for K.T** than K.D*. The rate constant of the hydrolysis step k3 is 6-7 s-1. The fluorescence decreases after formation of K.T** at a rate of 7-10 s-1. This change could occur in step 3 or in step 4 if k4 much greater than k3. The value of k4 is larger than 0.1 s-1. The steady state rate is 0.003 s-1 which agrees with the rate of ADP dissociation (k5). Step 5 is rate limiting in the scheme in agreement with the conclusion of Hackney (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318) that ADP dissociation is the rate-limiting step.     

Temperature dependence of force, velocity, and processivity of single kinesin molecules

Using the bead assay in optical microscopy equipped with optical tweezers, we have examined the effect of temperature on the gliding velocity, force, and processivity of single kinesin molecules interacting with a microtubule between 15 and 35 degrees C. The gliding velocity increased with the Arrhenius activation energy of 50 kJ/mol, consistent with the temperature dependence of the microtubule-dependent ATPase activity. Also, the average run length, i.e., a measure of processivity of kinesin, increased on increasing temperature. On the other hand, the generated force was independent of temperature, 7.34 +/- 0.33 pN (average +/- S.D., n = 70). The gliding velocities decreased almost linearly with an increase in force irrespective of temperature, implying that the efficiency of mechano-chemical energy conversion is maintained constant in this temperature range. Thus, we suggest that the force generation is attributable to the temperature-insensitive nucleotide-binding state(s) and/or conformational change(s) of kinesin-microtubule complex, whereas the gliding velocity is determined by the ATPase rate.     

The conformational cycle of kinesin

The stepping mechanism of kinesin can be thought of as a programme of conformational changes. We briefly review protein chemical, electron microscopic and transient kinetic evidence for conformational changes, and working from this evidence, outline a model for the mechanism. In the model, both kinesin heads initially trap Mg x ADP. Microtubule binding releases ADP from one head only (the trailing head). Subsequent ATP binding and hydrolysis by the trailing head progressively accelerate attachment of the leading head, by positioning it closer to its next site. Once attached, the leading head releases its ADP and exerts a sustained pull on the trailing head. The rate of closure of the molecular gate which traps ADP on the trailing head governs its detachment rate. A speculative but crucial coordinating feature is that this rate is strain sensitive, slowing down under negative strain and accelerating under positive strain.     

Alternating site ATPase pathway of rat conventional kinesin

The pathway of ATP hydrolysis by rat kinesin was established by pre-steady-state kinetic methods. A 406-residue long N-terminal fragment was shown by sedimentation equilibrium analysis to form a dimer with a K(d) of 46 nm. The pathway of ATP hydrolysis follows the Gilbert-Johnson pathway determined previously for a similarsized N-terminal fragment of Drosophila conventional kinesin. However, the rates of ADP release were at least 3-fold faster, and ATP hydrolysis was approximately 5-fold faster. Paralleling our previous mechanistic data, these results support an alternating site ATPase pathway, including a captive head state as an intermediate in the kinesin ATPase cycle. The kinetic data presented in this report once again point to the importance of the captive head state and argue against a pathway that short-circuits this key intermediate. In addition, several unique aspects of the rat kinesin kinetics reveal new aspects of the ATPase-coupling mechanism. These studies provide a baseline set of kinetic parameters against which future studies of rat kinesin mutants may be evaluated and directly correlated with the structure of the dimeric kinesin.     

ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility

Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed presteady-state kinetic and single molecule motility analyses on three of these mutants [Y138A, loop 11 triple (L248A/D249A/E250A), and E311A] that exhibited a similar approximately 3-fold reduction in both microtubule gliding velocity and microtubule-stimulated ATPase activity. All mutants showed normal second-order ATP binding kinetics, indicating correct folding of the active site. The Y138A and loop 11 triple mutants were defective both in nucleotide hydrolysis and in microtubule-stimulated ADP release rates, the latter suggesting a defect in allosteric communication between the microtubule and the active site. A single molecule fluorescence assay further revealed that the loop 11 mutant is defective in initiating processive motion, suggesting that this loop is important for the initial contact between kinesin and the microtubule. Y138A, on the other hand, can bind to the microtubule normally but cannot move processively. For E311A, neither the rate of nucleotide hydrolysis nor ADP release could account for its slower ATPase and gliding velocity, which suggests that either phosphate release or a conformational transition is rate-limiting in this mutant. The single molecule assay showed that E311A has a reduced velocity of movement, but is not defective in processivity. Thus, while these mutants behave similarly in solution ATPase and multiple motor gliding assays, kinetic and single molecule analyses reveal defects in distinct processes in kinesin's mechanochemical cycle.     

A structural pathway for activation of the kinesin motor ATPase

Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament-binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule-activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild-type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water-mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPASE: The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules.     

Fast backward steps and detachment of single kinesin molecules measured under a wide range of loads

To understand force generation under a wide range of loads, the stepping of single kinesin molecules was measured at loads from −20 to 42 pN by optical tweezers with high temporal resolution. The optical trap has been improved to halve positional noise and increase bandwidth by using 200-nm beads. The step size of the forward and backward steps was 8.2 nm even over a wide range of loads. Histograms of the dwell times of backward steps and detachment fit well to two independent exponential equations with fast (~0.4 ms) and slow (>3 ms) time constants, indicating the existence of a fast step in addition to the conventional slow step. The dwell times of the fast steps were almost independent of the load and ATP concentration, while those of the slow backward steps and detachment depended on those. We constructed the kinetic model to explain the fast and slow steps under a wide range of loads.     

Photocontrol of kinesin ATPase activity using an azobenzene derivative

Azobenzene is a photochromic molecule that undergoes rapid and reversible isomerization between the cis- and trans-forms in response to ultraviolet (UV) and visible (VIS) light irradiation, respectively. Here, we introduced the sulfhydryl-reactive azobenzene derivative 4-phenylazophenyl maleimide (PAM) into the functional region of kinesin to reversibly regulate the ATPase activity of kinesin by photoirradiation. We prepared five kinesin motor domain mutants, A247C, L249C, A252C, G272C and S275C, which contained a single reactive cysteine residue in loops L11 and L12. These loops are considered to be key regions for the functioning of kinesin as a motor protein. PAM was stoichiometrically incorporated into the cysteine residues in the loops of the mutants. The PAM-modified S275C mutant exhibited reversible alterations in ATPase activity accompanied by cis-trans isomerization upon UV and VIS light irradiation. The ATPase activity exhibited by the cis-isomer of the PAM bound to the mutant was two times higher than that of the trans-isomer. Further, the PAM-modified L249C mutant exhibited reversible alterations in ATPase activity on UV-VIS light irradiation but exhibited the opposite effect on UV and VIS light irradiation. Using a photochromic azobenzene derivative, we have demonstrated that the ATPase activity of the motor protein kinesin is photoregulated.     

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 (k_cat), whereas mutation in the β‐tubulin‐binding site lowers the affinity for MTs (K_0.5MT). 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.     

Regulation of KinI kinesin ATPase activity by binding to the microtubule lattice

KinI kinesins are important in regulating the complex dynamics of the microtubule cytoskeleton. They are unusual in that they depolymerize, rather than move along microtubules. To determine the attributes of KinIs that distinguish them from translocating kinesins, we examined the ATPase activity, microtubule affinity, and three-dimensional microtubule-bound structure of a minimal KinI motor domain. Together, the kinetic, affinity, and structural data lead to the conclusion that on binding to the microtubule lattice, KinIs release ADP and enter a stable, low-affinity, regulated state, from which they do not readily progress through the ATPase cycle. This state may favor detachment, or diffusion of the KinI to its site of action, the microtubule ends. Unlike conventional translocating kinesins, which are microtubule lattice-stimulated ATPases, it seems that with KinIs, nucleotide-mediated modulation of tubulin affinity is only possible when it is coupled to protofilament deformation. This provides an elegant mechanistic basis for their unique depolymerizing activity.     

Mechanical and chemical properties of cysteine-modified kinesin molecules.

To probe the structural changes within kinesin molecules, we made the mutants of motor domains of two-headed kinesin (4-411 aa) in which either all the five cysteines or all except Cys45 were mutated. A residual cysteine (Cys45) of the kinesin mutant was labeled with an environment-sensitive fluorescent probe, acrylodan. ATPase activity, mechanical properties, and fluorescence intensity of the mutants were measured. Upon acrylodan-labeled kinesin binding to microtubules in the presence of 1 mM AMPPNP, the peak intensity was enhanced by 3.4-fold, indicating the structural change of the kinesin head by the binding. Substitution of cysteines decreased both the maximum microtubule-activated ATPase and the sliding velocity to the same extent. However, the maximum force and the step size were not affected; the force produced by a single molecule was 6-6.5 pN, and a step size due to the hydrolysis of one ATP molecule by kinesin molecules was about 10 nm for all kinesins. This step size was close to a unitary step size of 8 nm. Thus, the mechanical events of kinesin are tightly coupled with the chemical events.     

The ATPase cross-bridge cycle of the Kar3 motor domain. Implications for single head motility

Kar3 is a minus-end directed microtubule motor involved in meiosis and mitosis in Saccharomyces cerevisae. Unlike Drosophila Ncd, the other well characterized minus-end directed motor that is a homodimer, Kar3 is a heterodimer with a single motor domain and either the associated polypeptides Cik1 or Vik1. Our mechanistic studies with Ncd showed that both motor domains were required for ATP-dependent motor domain detachment from the microtubule. We have initiated a series of experiments to compare the mechanistic requirements for Kar3 motility in direct comparison to Ncd. The results presented here show that the single motor domain of Kar3 (Met(383)-Lys(729)) exhibits characteristics similar to monomeric Ncd. The microtubule-activated steady-state ATPase cycle of Kar3 (k(cat) = 0.5 s(-1)) is limited by ADP release (0.4 s(-1)). Like monomeric Ncd, Kar3 does not readily detach from the microtubule with the addition of MgATP. These results show that the single motor domain of Kar3 is not sufficient for ATP-dependent microtubule dissociation, suggesting that structural elements outside of the catalytic core are required for the cyclic interactions with the microtubule for force generation.     

Effect of temperature on kinesin-driven microtubule gliding and kinesin ATPase activity

DeCuevas et al. [J. Cell Biol. 116 (1992) 957-965] demonstrated by circular dichroism spectroscopy for the kinesin stalk fragment that shifting temperature from 25 to 30 degrees C caused a conformational transition. To gain insight into functional consequences of such a transition, we studied the temperature dependence of a full-length kinesin by measuring both the velocity of microtubule gliding across kinesin-coated surfaces and microtubule-promoted kinesin ATPase activity in solution. The corresponding Arrhenius plots revealed distinct breaks at 27 degrees C, corroborating the temperature-dependent conformational transition for a motility-competent full-length kinesin. Microtubules were found to glide up to 45 degrees C; at higher temperatures, kinesin was irreversibly damaged.