Whereas kinesin I is designed to transport cargoes long distances in isolation, a closely related kinesin motor, Eg5, is designed to generate a sustained opposing force necessary for proper mitotic spindle formation. Do the very different roles for these evolutionarily related motors translate into differences in how they generate movement? We have addressed this question by examining when in the ATPase cycle the Eg5 motor domain and neck linker move through the use of a series of novel spectroscopic probes utilizing fluorescence resonance energy transfer, and we have compared our results to kinesin I. Our results are consistent with a model in which movement in Eg5 occurs in two sequential steps, an ATP-dependent docking of the neck linker, followed by a rotation or "rolling" of the entire motor domain on the microtubule surface that occurs with ATP hydrolysis. These two forms of movement are consistent with the functions of a motor designed to generate sustained opposing force, and hence, our findings support the argument that the mechanochemical features of a molecular motor are shaped more by the demands placed on it than by its particular family of origin.The last decade has witnessed a marked advance in our understanding of how molecular motors generate force and movement (1-6). Studies of both myosins and kinesins have revealed a variety of conserved structural elements that play key roles in mechanochemical transduction. These include switch I, switch II, and the P loop located within the catalytic site (3,5,6). Movements of these elements during ATP binding, hydrolysis, and product release lead to a series of conformational changes that are transmitted ultimately to the "business end" of the motor, the mechanical element that produces force and movement. In kinesin I, this mechanical element consists of an extended peptide sequence with variable conformation and flexibility, called the "neck linker" (2, 3). Spectroscopic and kinetic studies have led to a convincing model in which the neck linker assumes a random coil in the absence of nucleotide. ATP binding to the active site causes the neck linker to dock along a hydrophobic surface in the motor (7-10). This process, which is very rapid (Ͼ800 s Ϫ1 ) at room temperature, immobilizes the neck linker and effectively "throws" the tethered head of a kinesin dimer forward, toward the next tubulin-docking site (11,12,25). Variable flexibility and ATP-induced docking are features of the kinesin I neck linker that are well suited to the physiologic role of this motor as a transport engine. Variable flexibility in the neck linker of the tethered head allows it to undergo a diffusive search for the next microtubule-docking site, whereas ATP-induced docking of the neck linker of the attached head helps position the tethered head in a forward position and reduces the probability of backward stepping (10,(13)(14)(15)(16). Thus, the physiologic requirements placed on a transport motor like kinesin I translate into structural features of its mechanical element that s...

Recent models of the kinesin mechanochemical cycle provide some conflicting information on how the neck linker contributes to movement. Some spectroscopic approaches suggest a nucleotide-induced order-to-disorder transition in the neck linker. However, cryoelectron microscopic imaging suggests instead that nucleotide alters the orientation of the neck linker when docked on the microtubule surface. Furthermore, since these studies utilized transition state or non-hydrolyzable nucleotide analogs, it is not clear at what point in the ATPase cycle this reorientation of the neck linker occurs. We have addressed this issue by developing a strategy to examine the effect of nucleotide on the orientation of the neck linker based on the technique of fluorescence resonance energy transfer. Transient kinetic studies utilizing this approach support a model in which ATP binding leads to two sequential isomerizations, the second of which reorients the neck linker in relation to the microtubule surface.Models of how molecular motors utilize energy from ATP hydrolysis to generate force or movement are still under active investigation. In the case of myosin, a variety of experimental results suggest that an isomerization associated with or following phosphate release leads to a swinging of a rigid lever arm contained within the regulatory domain (1, 2). However, additional rotation of this lever arm may occur with ADP release in at least some myosin isoforms (3-5). In kinesin motors, the situation is even less clear. In the case of conventional kinesin, a carboxyl-terminal region of the motor domain, referred to as the "neck linker," has been proposed to alter its orientation during the course of ATP binding and/or hydrolysis (6). Studies with spectroscopic and electron-dense labels have led to a model in which the neck linker alternates between an ordered orientation, in which it is docked on the motor domain, and one that is disordered (7). This order-to-disorder transition has been proposed to be dependent on the nature of the nucleotide in the active site. It is the basis for a recently proposed model in which cycles of docking and release of the neck linker cause the unattached kinesin head to find the next binding site on the microtubule (7).However, several lines of evidence suggest that this model may not completely describe the mechanism of processive motility for kinesin. First, cryoelectron microscopy of gold-labeled, kinesin-decorated microtubules showed that in rigor and ADP, the neck linker assumes two distinct orientations, separate from that seen with ATP analogs (7). This suggests the presence of three discrete kinesin states, one induced by ATP and two by ADP or rigor, which is in fact consistent with recent kinetic studies of kinesin motor domain mutants (8). Second, it is not clear at what point in the ATPase cycle the change in state of the neck linker occurs. Both AMP-PNP 1 and ADP ϩ aluminum fluoride immobilize the neck linker and appear to dock it onto the motor domain. However, AMP-PNP is thought to mimic...

The ability of kinesin to travel long distances on its microtubule track without dissociating has led to a variety of models to explain how this remarkable degree of processivity is maintained. All of these require that the two motor domains remain enzymatically "out of phase," a behavior that would ensure that, at any given time, one motor is strongly attached to the microtubule. The maintenance of this coordination over many mechanochemical cycles has never been explained, because key steps in the cycle could not be directly observed. We have addressed this issue by applying several novel spectroscopic approaches to monitor motor dissociation, phosphate release, and nucleotide binding during processive movement by a dimeric kinesin construct. Our data argue that the major effect of the internal strain generated when both motor domains of kinesin bind the microtubule is to block ATP from binding to the leading motor. This effect guarantees the two motor domains remain out of phase for many mechanochemical cycles and provides an efficient and adaptable mechanism for the maintenance of processive movement.Members of the kinesin family of molecular motors are capable of taking over 100 steps on their microtubule track without dissociating, a feature that would be necessary for a transport motor that operates in isolation (1-6). A variety of kinetic, structural, and mechanical studies have revealed that this processive behavior requires that two motors of kinesin remain in different structural and enzymatic states during a processive run (7-10, 11, 12). This would ensure that, at any given time, at least one of the two heads would remain strongly attached to its track, preventing the motor from prematurely detaching. Such coordination requires a way for the two motor domains to communicate their structural states to each other while walking processively. Several lines of evidence suggest that this allosteric communication is mediated through the internal load generated when both heads attach to the microtubule (1, 13-16). As illustrated below in Fig. 1, kinesin initiates its mechanochemical cycle with its attached head (green) nucleotide free and its tethered head (magenta) containing ADP in the active site. ATP binding to the attached head reorients its neck linker (blue), which swings the tethered head forward to the next tubulin-docking site. ADP is then released from the new, weakly bound leading head (magenta) to produce an intermediate in which both heads are strongly bound to the microtubule. This situation would generate rearward strain on the neck linker of the leading head, depicted as a left pointing arrow, and forward strain on the corresponding structure of the trailing head, depicted as a right pointing arrow.It has been proposed that this strain generates processivity by accelerating release of the trailing head (13,17). In this mechanism, release of the ADP-containing trailing head would be very slow in the absence of forward strain and fast in its presence. In such a system, the greater that forwar...

A variety of models have recently emerged to explain how the molecular motor kinesin is able to maintain processive movement for over 100 steps. Although these models differ in significant features, they all predict that kinesin's catalytic domains intermittently separate from each other as the motor takes 8-nm steps along the microtubule. Furthermore, at some point in this process, one molecule of ATP is hydrolyzed per step. However, exactly when hydrolysis and product release occur in relation to this forward step have not been established. Furthermore, the rate at which this separation occurs as well as the speed of motor stepping onto and release from the microtubule have not been measured. In the absence of this information, it is difficult to critically evaluate competing models of kinesin function. We have addressed this issue by developing spectroscopic probes whose fluorescence is sensitive to motormotor separation or microtubule binding. The kinetics of these fluorescence changes allow us to directly measure how fast kinesin steps onto and releases from the microtubule and provide insight into how processive movement is maintained by this motor.Several features of kinesin's mechanochemistry contribute to its ability to move long distances on the microtubule without dissociating. These include its high duty ratio and ability to hydrolyze multiple ATP molecules per force productive encounter-features that enhance the probability that a motor will remain attached even under load (1-6). However, processive movement also requires two heads, and coordination between these two motor domains is essential if kinesin is to move in an orderly fashion along its track (7,8). Two models have recently appeared to explain how the motor domains work together to bring about processive movement. In the hand-over-hand model (1, 9), each head alternates between a forward and rearward orientation on the microtubule, whereas in the inchworm model (10), the forward and rearward heads retain their relative positions throughout the mechanochemical cycle. The hand-over-hand model proposes that ATP binding docks the neck linker of the attached head, and this swings the tethered head forward toward the next microtubule ␤ subunit 8 nm away (11,12). ATP hydrolysis on the rear head and ADP dissociation from the newly attached forward head follow, and they lead to dissociation of the rearward head from the microtubule. The net effect is hydrolysis of one ATP molecule per 8-nm step in the plus end direction (11, 13). In the inchworm model, ATP binding, hydrolysis, and dissociation of ADP are associated with alternating separation and association of the two heads, only one of which is enzymatically active. However, precisely where nucleotide binding and hydrolysis fit into the overall scheme has not been determined (10).Despite their differences, both models agree on several key features. First, ATP induces the two heads to separate by 8 nm, as one of them steps forward in the "ϩ" direction. Second, at some point in the cycle, ATP is hy...