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.     

Moving a microtubule may require two heads: a kinetic investigation of monomeric Ncd

Ncd is a minus-end-directed microtubule motor and a member of the kinesin superfamily. The Ncd dimer contains two motor domains, and cooperative interactions between the heads influence the interactions of each respective motor domain with the microtubule. The approach we have taken to understand the cooperativity between the two motor domains is to analyze the ATPase cycle of dimeric MC1 and monomeric MC6. The steps in the ATPase cycle where cooperativity occurs can be identified by comparing the two mechanisms. The rate-limiting step in the MC6 mechanism is ADP release at 3.4 s(-)(1). The observed rate constant for ATP-induced dissociation from the microtubule is 14 s(-)(1). However, the relative amplitude associated with MC6 dissociation is extremely small in comparison to the amplitude associated with dimeric MC1 dissociation kinetics. The amplitude data indicate that monomeric MC6 does not detach from the microtubule during the initial turnovers of ATP, and ATP hydrolysis is uncoupled from movement. The results show that cooperative interactions between the motor domains of the dimer are required for ATP-dependent dissociation; therefore, one function of the partner motor domain may be to weaken the interaction of the adjacent head with the microtubule.     

Kinetic studies of dimeric Ncd: evidence that Ncd is not processive

Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules. The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein [Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. (1993) J. Biol. Chem. 268, 9005-9013]. Acid chemical quench flow methods were used to measure directly the rate of ATP hydrolysis, and stopped-flow kinetic methods were used to determine the kinetics of mantATP binding, mantADP release, dissociation of MC1 from the microtubule, and binding of MC1 to the microtubule. The results define a minimal kinetic mechanism, M.N + ATP M.N.ATP M.N.ADP.P N. ADP.P N.ADP + P M.N.ADP M.N + ADP, where N, M, and P represent Ncd, microtubules, and inorganic phosphate respectively, with k(+1) = 2.3 microM(-1) s(-1), k(+2) =23 s(-1), k(+3) =13 s(-1), k(+5)= 0.7 microM(-)(1) s(-)(1), and k(+6) = 3.7 s(-)(1). Phosphate release (k(+4)) was not measured directly although it is assumed to be fast relative to ADP release because Ncd is purified with ADP tightly bound at the active site. ATP hydrolysis occurs at 23 s(-)(1) prior to Ncd dissociation at 13 s(-)(1). The pathway for ATP-promoted detachment (steps 1-3) of Ncd from the microtubule is comparable to kinesin's. However, there are two major differences between the mechanisms of Ncd and kinesin. In contrast to kinesin, mantADP release for Ncd at 3.7 s(-)(1) is the slowest step in the pathway and is believed to limit steady-state turnover. Additionally, the burst amplitude observed in the pre-steady-state acid quench experiments is stoichiometric, indicating that Ncd, in contrast to kinesin, is not processive for ATP hydrolysis.     

The Origin of Minus-end Directionality and Mechanochemistry of Ncd Motors

Adaptation of molecular structure to the ligand chemistry and interaction with the cytoskeletal filament are key to understanding the mechanochemistry of molecular motors. Despite the striking structural similarity with kinesin-1, which moves towards plus-end, Ncd motors exhibit minus-end directionality on microtubules (MTs). Here, by employing a structure-based model of protein folding, we show that a simple repositioning of the neck-helix makes the dynamics of Ncd non-processive and minus-end directed as opposed to kinesin-1. Our computational model shows that Ncd in solution can have both symmetric and asymmetric conformations with disparate ADP binding affinity, also revealing that there is a strong correlation between distortion of motor head and decrease in ADP binding affinity in the asymmetric state. The nucleotide (NT) free-ADP (φ-ADP) state bound to MTs favors the symmetric conformation whose coiled-coil stalk points to the plus-end. Upon ATP binding, an enhanced flexibility near the head-neck junction region, which we have identified as the important structural element for directional motility, leads to reorienting the coiled-coil stalk towards the minus-end by stabilizing the asymmetric conformation. The minus-end directionality of the Ncd motor is a remarkable example that demonstrates how motor proteins in the kinesin superfamily diversify their functions by simply rearranging the structural elements peripheral to the catalytic motor head domain.     

Kinetics processivity and the direction of motion of Ncd.

The kinetic mechanism of the nonclaret disjunctional protein (Ncd) motor was investigated using the dimer termed MC1 (residues 209-700), which has been shown to exhibit negative-end directed motility (Chandra et al., 1993). The kinetic properties are similar to those of the monomeric Ncd motor domain (Pechatnikova and Taylor, 1997). The maximum steady-state ATPase activity of 1.5 s(-1) is half as large as for the monomeric motor. Dissociation constants in the presence of nucleotides showed the same trend but with approximately a two-fold decrease in the values: K(d) values are 1.0 microM for ADP-AlF(4), 1.1 microM for ATPgammaS, 1.5 microM for ATP, 3 microM for ADP, and 10 microM for ADP-vanadate (in 25 mM NaCl, 22 degrees C). The apparent second-order rate constants for the binding of ATP and ADP to the microtubule-motor complex (MtMC1) are 2 microM(-1) s(-1). Based on measurements at high microtubule concentrations the kinetic steps were fitted to the scheme,[see text] where N refers to one head of the dimer and T, D, and P stand for ATP, ADP, and inorganic phosphate. k(1) and k(-4) are the first-order rate constants of the transition induced by the binding of mant ATP and mant ADP respectively. ADP release is the main rate-limiting step in the MtMC1 mechanism. The binding of the MC1-mant ADP complex to microtubules released less than half of the mant ADP (alternating site reactivity). The second mant ADP is only released by the binding of nucleotides that dissociate the MtMC1 complex (ATP and ADP but not AMPPNP). The apparent rate constant for dissociation of the second mant ADP is four times smaller than the first and much smaller than the rate of dissociation of MtMC1 by ATP or ADP. These results are explained by a model in which MC1.ADP is first dissociated from the microtubule by ATP, followed by rebinding to the microtubule by the ADP-containing head. Ncd may follow a different reaction pathway than does kinesin, but the differences in rate constants do not explain the opposite direction of motion. The kinetic evidence and the high ratio of motile velocity to ATPase support a nonprocessive, low duty cycle mechanism for the Ncd motor.     

Two-state displacement by the kinesin-14 Ncd stalk

The nonprocessive kinesin-14 Ncd motor binds to microtubules and hydrolyzes ATP, undergoing a single displacement before releasing the microtubule. A lever-like rotation of the coiled-coil stalk is thought to drive Ncd displacements or steps along microtubules. Crystal structures and cryoelectron microscopy reconstructions imply that stalk rotation is correlated with ADP release and microtubule binding by the motor. Here we report FRET assays showing that the end of the stalk is more than ~9nm from the microtubule when wild-type Ncd binds microtubules without added nucleotide, but the stalk is within ~6nm of the microtubule surface when the microtubule-bound motor binds an ATP analogue, matching the rotated state observed in crystal structures. We propose that the stalk rotation is initiated when the motor binds to microtubules and releases ADP, and is completed when ATP binds.     

Getting in sync with dimeric Eg5. Initiation and regulation of the processive run

Eg5/KSP is the kinesin-related motor protein that generates the major plus-end directed force for mitotic spindle assembly and dynamics. Recent work using a dimeric form of Eg5 has found it to be a processive motor; however, its mechanochemical cycle is different from that of conventional Kinesin-1. Dimeric Eg5 appears to undergo a conformational change shortly after collision with the microtubule that primes the motor for its characteristically short processive runs. To better understand this conformational change as well as head-head communication during processive stepping, equilibrium and transient kinetic approaches have been used. By contrast to the mechanism of Kinesin-1, microtubule association triggers ADP release from both motor domains of Eg5. One motor domain releases ADP rapidly, whereas ADP release from the other occurs after a slow conformational change at approximately 1 s(-1). Therefore, dimeric Eg5 begins its processive run with both motor domains associated with the microtubule and in the nucleotide-free state. During processive stepping however, ATP binding and potentially ATP hydrolysis signals rearward head advancement 16 nm forward to the next microtubule-binding site. This alternating cycle of processive stepping is proposed to terminate after a few steps because the head-head communication does not sufficiently control the timing to prevent both motor domains from entering the ADP-bound state simultaneously.     

Visualizing a new binding site of ncd-motor domain on tubulin

Ncd is a microtubule minus-end directed motor of the kinesin superfamily. Previously it has been shown that ncd and kinesin motor domains share the same major binding site on microtubules. Here we report a three-dimensional EM reconstruction of negatively stained two-dimensional Zn-induced tubulin crystal sheets (Zn-sheets) decorated with the ncd motor domain at a resolution of 16 A. This work has revealed a second specific binding site for the ncd motor domain. The motor binding site on the tubulin Zn-sheets spans both alpha and beta tubulin subunits. This binding site is located at a position different from the previously identified ncd binding site on microtubules and may play a role in motor function.     

The role of ATP hydrolysis for kinesin processivity

Conventional kinesin is a highly processive, plus-end-directed microtubule-based motor that drives membranous organelles toward the synapse in neurons. Although recent structural, biochemical, and mechanical measurements are beginning to converge into a common view of how kinesin converts the energy from ATP turnover into motion, it remains difficult to dissect experimentally the intermolecular domain cooperativity required for kinesin processivity. We report here our pre-steady-state kinetic analysis of a kinesin switch I mutant at Arg(210) (NXXSSRSH, residues 205-212 in Drosophila kinesin). The results show that the R210A substitution results in a dimeric kinesin that is defective for ATP hydrolysis and a motor that cannot detach from the microtubule although ATP binding and microtubule association occur. We propose a mechanistic model in which ATP binding at head 1 leads to the plus-end-directed motion of the neck linker to position head 2 forward at the next microtubule binding site. However, ATP hydrolysis is required at head 1 to lock head 2 onto the microtubule in a tight binding state before head 1 dissociation from the microtubule. This mechanism optimizes forward movement and processivity by ensuring that one motor domain is tightly bound to the microtubule before the second can detach.     

Microscopic evidence for a minus-end-directed power stroke in the kinesin motor ncd

We used cryo-electron microscopy and image reconstruction to investigate the structure and microtubule-binding configurations of dimeric non-claret disjunctional (ncd) motor domains under various nucleotide conditions, and applied molecular docking using ncd's dimeric X-ray structure to generate a mechanistic model for force transduction. To visualize the alpha-helical coiled-coil neck better, we engineered an SH3 domain to the N-terminal end of our ncd construct (296-700). Ncd exhibits strikingly different nucleotide-dependent three-dimensional conformations and microtubule-binding patterns from those of conventional kinesin. In the absence of nucleotide, the neck adapts a configuration close to that found in the X-ray structure with stable interactions between the neck and motor core domain. Minus-end-directed movement is based mainly on two key events: (i) the stable neck-core interactions in ncd generate a binding geometry between motor and microtubule which places the motor ahead of its cargo in the minus-end direction; and (ii) after the uptake of ATP, the two heads rearrange their position relative to each other in a way that promotes a swing of the neck in the minus-end direction.     

A kinesin switch I arginine to lysine mutation rescues microtubule function

Switch I and II are key active site structural elements of kinesins, myosins, and G-proteins. Our analysis of a switch I mutant (R210A) in Drosophila melanogaster kinesin showed a reduction in microtubule affinity, a loss in cooperativity between the motor domains, and an ATP hydrolysis defect leading to aberrant detachment from the microtubule. To investigate the conserved arginine in switch I further, a lysine substitution mutant was generated. The R210K dimeric motor has lost the ability to hydrolyze ATP; however, it has rescued microtubule function. Our results show that R210K has restored microtubule association kinetics, microtubule affinity, ADP release kinetics, and motor domain cooperativity. Moreover, the active site at head 1 is able to distinguish ATP, ADP, and AMP-PNP to signal head 2 to bind the microtubule and release mantADP with kinetics comparable with wild-type. Therefore, the structural pathway of communication from head 1 to head 2 is restored, and head 2 can respond to this signal by binding the microtubule and releasing mantADP. Structural modeling revealed that lysine could retain some of the hydrogen bonds made by arginine but not all, suggesting a structural hypothesis for the ability of lysine to rescue microtubule function in the Arg210 mutant.     

Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern

Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αβ-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1-microtubule interaction and positioning the motor for the start of the powerstroke. The results indicate that head-head communication is mediated through the adjoining coiled coil.     

Mechanistic analysis of the Saccharomyces cerevisiae kinesin Kar3

Kar3 is a minus-end-directed microtubule motor that is implicated in meiotic and mitotic spindle function in Saccharomyces cerevisiae. To date, the only truncated protein of Kar3 that has been reported to promote unidirectional movement in vitro is GSTKar3. This motor contains an NH2-terminal glutathione S-transferase (GST) tag followed by the Kar3 sequence that is predicted to form an extended alpha-helical coiled-coil. The alpha-helical domain leads into the neck linker and COOH-terminal motor domain. Kar3 does not homodimerize with itself but forms a heterodimer with either Cik1 or Vik1, both of which are non-motor polypeptides. We evaluated the microtubule-GSTKar3 complex in comparison to the microtubule-Kar3 motor domain complex to determine the distinctive mechanistic features required for GSTKar3 motility. Our results indicate that ATP binding was significantly faster for GSTKar3 than that observed previously for the Kar3 motor domain. In addition, microtubule-activated ADP release resulted in an intermediate that bound ADP weakly in contrast to the Kar3 motor domain, suggesting that after ADP release, the microtubule-GSTKar3 motor binds ATP in preference to ADP. The kinetics also showed that GST-Kar3 readily detached from the microtubule rather than remaining bound for multiple ATP turnovers. These results indicate that the extended alpha-helical domain NH2-terminal to the catalytic core provides the structural transitions in response to the ATPase cycle that are critical for motility and that dimerization is not specifically required. This study provides the foundation to define the mechanistic contributions of Cik1 and Vik1 for Kar3 force generation and function in vivo.     

The Ncd tail domain promotes microtubule assembly and stability

Non-claret disjunctional (Ncd) is a Drosophila kinesin-like motor required for spindle assembly and maintenance in oocytes and early embryos. Ncd has an ATP-independent microtubule binding site in the N-terminal tail domain as well as an ATP-dependent microtubule binding site in the C-terminal motor domain. The Ncd tail domain shares many properties with the microtubule-associated proteins that regulate microtubule assembly, including microtubule binding and bundling activity and an abundance of basic and proline residues. Given these similarities, we examined the ability of Ncd tail domain proteins to promote MT assembly and stability. The results indicate that the Ncd tail domain can promote MT assembly and stabilize MTs against conditions that induce MT disassembly, and suggest that Ncd may influence MT dynamics within the spindle.     

Spatial relationship between heads of dimeric Ncd in the presence of nucleotides and microtubules

Kinesins are molecular motors that produce mechanical work at the expense of ATP hydrolysis. Here, we studied Ncd (non-claret disjunctional), a (-)-end-directed member of this superfamily. To gain insight into the mechanism by which Ncd generates force and movement, we measured distances between the heads in dimeric Ncd-250-700 using fluorescence resonance energy transfer (FRET). About 5% of Ncd heads were labeled with 1,5-IAEDANS (donor), and the remaining thiol groups were modified with QSY35-iodoacetamide (acceptor). Several lines of experimental evidence suggest that the probes were conjugated to Cys-670 in each head of the dimer. The measured donor-acceptor distance was about 35 A. Nucleotides (ADP, ATP, and AMP-PNP) in the presence and absence of microtubules had only small effects on the interhead distances. Similar results were obtained for bidirectional Ncd mutant in which Asn-340 was replaced by a lysine. The results argue against models of Ncd movement in which the heads undergo large spatial rearrangements during mechanochemical cycle and suggest Gly-347 as a possible pivot point for the head rotation.     

Reversing a ‘backwards’ motor

Ncd, a kinesin-related microtubule motor protein that moves the ‘wrong’ way on microtubules, towards the minus ends, has now been made to move like kinesin, towards plus ends, by fusing regions from outside the kinesin motor domain to the Ncd motor.^1,2 Since it is the kinesin motor domain that binds to and moves on the microtubule, the finding that regions outside the motor domain can confer directionality of Ncd movement is remarkable—it implies a structural basis for motor polarity. Here we consider this finding from a structural point of view and discuss the implications for motor function and evolution. BioEssays 20:108–112, 1998. © 1998 John Wiley & Sons, Inc.     

Coupled chemical and mechanical reaction steps in a processive Neurospora kinesin.

We show using single molecule optical trapping and transient kinetics that the unusually fast Neurospora kinesin is mechanically processive, and we investigate the coupling between ATP turnover and the mechanical actions of the motor. Beads carrying single two-headed Neurospora kinesin molecules move in discrete 8 nm steps, and stall at approximately 5 pN of retroactive force. Using microtubule-activated release of the fluorescent analogue 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mantADP) to report microtubule binding, we found that initially only one of the two motor heads binds, and that the binding of the other requires a nucleotide 'chase'. mantADP was released from the second head at 4 s(-1) by an ADP chase, 5 s(-1) by 5'-adenylylimidodiphosphate (AMPPNP), 27 s(-1) by ATPgammaS and 60 s(-1) by ATP. We infer a coordination mechanism for molecular walking, in which ATP hydrolysis on the trailing head accelerates leading head binding at least 15-fold, and leading head binding then accelerates trailing head unbinding at least 6-fold.     

Are Coiled-Coils of Dimeric Kinesins Unwound during Their Walking on Microtubule?

Dimeric kinesin motor proteins such as homodimeric kinesin-1, homodimeric Ncd and heterodimeric Kar3/Vik1are composed of two head domains which are connected together by a rod-shaped, coiled-coil stalk. Despite the extensive and intensive studies on structures, kinetics, dynamics and walking mechanism of the dimers, whether their coiled-coils are unwound or not during their walking on the microtubule is still an unclear issue. Here, we try to clarify this issue by using molecular dynamics simulations. Our simulation results showed that, for Ncd, a large change in potential of mean force is required to unwind the coiled-coil by only several pairs of residues. For both Ncd and kinesin-1, the force required to initiate the coiled-coil unwinding is larger than that required for unfolding of the single [Formula: see text]-helix that forms the coiled-coil or is larger than that required to unwind the DNA duplex, which is higher than the unbinding force of the kinesin head from the microtubule in strong microtubule-binding states. Based on these results and the comparison of the sequence between the coiled-coil of Kar3/Vik1 and those of Ncd and kinesin-1, it was deduced that the coiled-coil of the Kar3/Vik1 should also be very stable. Thus, we concluded that the coiled-coils of kinesin-1, Ncd and Kar3/Vik1 are almost impossible to unwind during their walking on the microtubule.     

Arabidopsis thaliana protein,ATK1, is a minus-end directed kinesin that exhibits non-processive movement

The microtubule cytoskeleton forms the scaffolding of the meiotic spindle. Kinesins, which bind to microtubules and generate force via ATP hydrolysis, are also thought to play a critical role in spindle assembly, maintenance, and function. The A. thaliana protein, ATK1 (formerly known as KATA), is a member of the kinesin family based on sequence similarity and is implicated in spindle assembly and/or maintenance. Thus, we want to determine if ATK1 behaves as a kinesin in vitro, and if so, determine the directionality of the motor activity and processivity character (the relationship between molecular "steps" and microtubule association). The results show that ATK1 supports microtubule movement in an ATP-dependent manner and has a minus-end directed polarity. Furthermore, ATK1 exhibits non-processive movement along the microtubule and likely requires at least four ATK1 motors bound to the microtubule to support movement. Based on these results and previous data, we conclude that ATK1 is a non-processive, minus-end directed kinesin that likely plays a role in generating forces in the spindle during meiosis.     

Motor domain mutation traps kinesin as a microtubule rigor complex

Conventional kinesin is a highly processive, microtubule-based motor protein that drives the movement of membranous organelles in neurons. Using in vivo genetics in Drosophila melanogaster, Glu164 was identified as an amino acid critical for kinesin function [Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514]. Glu164 is located at the beta-strand 5a/loop 8b junction of the catalytic core and projects toward the microtubule binding face in close proximity to key residues on beta-tubulin helix alpha12. Substitution of Glu(164) with alanine (E164A) results in a dimeric kinesin with a dramatic reduction in the microtubule-activated steady-state ATPase (5 s(-1) per site versus 22 s(-1) per site for wild-type). Our analysis shows that E164A binds ATP and microtubules with a higher affinity than wild-type kinesin. The rapid quench and stopped-flow results provide evidence that ATP hydrolysis is significantly faster and the precise coordination between the motor domains is disrupted. The data reveal an E164A intermediate that is stalled on the microtubule and cannot bind and hydrolyze ATP at the second head.