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. 2015 Jun 11;24(7):1047–1056. doi: 10.1002/pro.2697

Kinesin, 30 years later: Recent insights from structural studies

Weiyi Wang 1,2, Luyan Cao 2, Chunguang Wang 1, Benoît Gigant 2, Marcel Knossow 2,*
PMCID: PMC4500306  PMID: 25975756

Abstract

Motile kinesins are motor proteins that move unidirectionally along microtubules as they hydrolyze ATP. They share a conserved motor domain (head) which harbors both the ATP- and microtubule-binding activities. The kinesin that has been studied most moves toward the microtubule (+)-end by alternately advancing its two heads along a single protofilament. This kinesin is the subject of this review. Its movement is associated to alternate conformations of a peptide, the neck linker, at the C-terminal end of the motor domain. Recent progress in the understanding of its structural mechanism has been made possible by high-resolution studies, by cryo electron microscopy and X-ray crystallography, of complexes of the motor domain with its track protein, tubulin. These studies clarified the structural changes that occur as ATP binds to a nucleotide-free microtubule-bound kinesin, initiating each mechanical step. As ATP binds to a head, it triggers orientation changes in three rigid motor subdomains, leading the neck linker to dock onto the motor core, which directs the other head toward the microtubule (+)-end. The relationship between neck linker docking and the orientations of the motor subdomains also accounts for kinesin's processivity, which is remarkable as this motor protein only falls off from a microtubule after taking about a hundred steps. As tools are now available to determine high-resolution structures of motor domains complexed to their track protein, it should become possible to extend these studies to other kinesins and relate their sequence variations to their diverse properties.

Keywords: motor protein, structure, microtubules, mechanism, processivity, myosin

Introduction

Motile kinesins are motor proteins that move unidirectionally along microtubules as they hydrolyze ATP. The first kinesin was discovered 30 years ago1,2; since then many more were identified, often based on the sequence conservation of their motor domain (or head). Kinesins have been classified according to the position of the motor domain in the polypeptide chain (N-terminal, Internal, or C-terminal), to their polypeptide chain composition (monomers, homodimers, heterodimers…) and to their directionality (moving toward the (+)-end of microtubules or toward the (-)-end). A standardized classification of kinesins into 14 classes is now commonly used.3 Most kinesins are motile, with the notable exception of subfamily 13 members, which have an internally localized motor domain and are out of the focus of this review. Kinesins that move toward the (+)-end of microtubules are the most numerous. The most studied one was initially named conventional kinesin and is hereafter called kinesin. It belongs to the kinesin-1 class. Its motor domain is at the N-terminal end of its polypeptide chain while the other end links to the cargo being transported. A flexible “neck” region and a coiled coil connect the two ends and hold them together. The structural mechanism of this kinesin is the main subject of this review.

In kinesins, the motor domain is the business end of the molecule: it harbors both the ATP- and microtubule-binding activities. During motility, kinesin takes eight nanometer steps by alternately advancing each of its two heads, tracking along single protofilaments.4–7 Each motor domain cycles between conformations that are strongly attached to microtubules (nucleotide-free and ATP-bound) or weakly attached (ADP-bound).8 Several lines of evidence strongly suggest that movement is associated to alternate conformations of a ca 15 amino acid peptide at the C-terminal end of the motor domains.9–11 This peptide, called the neck linker, is either disordered or adopts a well defined structure as it docks onto the motor domain core, with its N- to C-terminal direction being oriented toward the microtubule (+)-end.

Insight into the kinesin mechanism was gained through structural studies. High-resolution crystallographic structures of isolated kinesin motor domains were initially determined.12–14 Their comparison with the structures of evolutionarily-related myosins led to the tentative identification of kinesin residues involved in ATP γ-phosphate binding, which was eventually confirmed when a structure of a kinesin motor domain bound to AMPPNP, a stable ATP analog, was determined.15 What remained unclear until recently is how the alternate occupancy of the two conformations of the neck linker is related to ATP binding and hydrolysis. This relation is precisely tuned as kinesin performs one step for each ATP it hydrolyzes.

Kinesins’ processivity has also attracted much attention. It depends very much on the motor considered but in some instances it becomes amazing. This is in particular the case of the kinesin that is the subject of this review, which performs about one hundred steps before detaching from the microtubule. This means that each motor domain in a dimer alternately detaches from the microtubule and rebinds to the same microtubule many times before the dimer falls off.16 Processivity has been shown to depend on the length of the neck linker,17 on the load applied to the kinesin and on its direction,18 but the structural mechanism that keeps one motor domain attached to a tubulin on the microtubule until the other motor domain of the dimer has rebound to the neighboring tubulin in the (+)-end direction has remained unclear.

Two other families of motor proteins, dyneins and myosins, move unidirectionally along cellular filaments (microtubules and microfilaments, respectively) as they hydrolyze ATP. These motors are much larger proteins than kinesins. Dyneins, which are the largest ones, have only been recently structurally studied and much remains to be discovered about their mechanism.19 Myosins, although of much smaller molecular weight than dyneins, are also much larger proteins than kinesins. Therefore, it was totally unexpected that these two families of proteins interact with the ATP γ-phosphate via a conserved motif.12 It remained nevertheless to be seen whether the similarities of the interaction with the nucleotide in these two families of motor proteins go beyond this.

Earlier work on the role of kinesins in intracellular transport,20 on the comparison of myosins and kinesins mechanisms,21,22 on kinesin motor mechanics23 and structural data on isolated kinesins24–26 have been previously reviewed. Here, we will consider recent advances in the structural mechanism of kinesins that have become possible because of the availability of high-resolution structures of kinesins bound to their track protein. The emphasis will mainly be on three areas: the precise relationship between ATP binding and movement, the structural mechanisms for processivity and the relationship between the mechanisms of myosins and kinesins. We will also briefly mention motile kinesins targeting inhibitors whose binding sites have been identified, suggesting a possible explanation of their mechanism. Finally, we will discuss areas where further progress in our understanding may soon become possible. But, before going into these issues, we will compare kinesin structures determined by cryo electron microscopy (cryo-EM) and X-ray crystallography. This comparison is particularly interesting because both methods have simultaneously, and therefore mostly independently, yielded an atomic model of the kinesin structural mechanism.

Kinesin structures have been determined by X-ray crystallography and cryo electron microscopy

As it moves along microtubules, kinesin binds ATP, hydrolyses it, and releases the reaction products (ADP and phosphate) before starting a new cycle. As this happens, the motor domain alternately binds to and unbinds from microtubules. There are therefore three main states along the nucleotide cycle (Fig. 1); structures have been determined for all of them. In particular, the structure of the motor domain in complex with tubulin was determined by X-ray crystallography27,28 and by high-resolution cryo-EM,29,30 in the nucleotide free state and when ATP-bound. The X-ray structure of kinesin-ATP bound to tubulin was used to determine the corresponding cryo-EM model30 so that these two structures are not really independently determined. Nucleotide-free kinesin was studied by both methods, and the results were reported simultaneously. The structures differ by their resolutions: 6 Å for cryo-EM and 2.2 Å for X-ray crystallography. They also differ because, in the crystal, tubulin had the conformation it adopts when it is isolated, whereas the structure determined by cryo-EM is that of nucleotide-free kinesin bound to a microtubule, its physiological partner. In a microtubule, tubulin is straight (its two subunits are related to each other by a translation), whereas it is curved otherwise (in addition to a translation, a ca. 10° rotation is required to relate the two subunits).31 Despite these different conformations of tubulin, once the two kinesin structures have been superimposed, the root mean square deviation (r.m.s.d.) between atoms in α helices or β sheets is 0.8 Å (over 157 Cαs, out of a total of 325). Therefore, the two kinesin structures are generally similar.

Figure 1.

Figure 1

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The kinesin nucleotide cycle. Neck linker docking, that occurs upon ATP binding to microtubule-bound kinesin is also presented.

Because the secondary structure is highly conserved in the X-ray and cryo-EM models, there are only two potential differences: the conformations of loops and those of the amino acid side chains. Loops differ substantially more than secondary structure elements. In the case of some loops, this may be for physical reasons: A contact in the crystal that obviously does not exist when kinesin is bound to a microtubule or a contact with tubulin that might differ because tubulin conformations differ in the crystal and in microtubules. Nevertheless, even when one considers loops that are not involved in any of these interactions, perhaps as a consequence of the limited cryo-EM resolution, their conformations differ more that those of secondary structure elements (r.m.s.d.: 1.4 Å over 75 Cαs). Concerning side chains, as they are not visualized in the kinesin cryo-EM maps,30 in the cryo-EM structure their positions result from modeling, albeit constrained by the structure of the polypeptide main chain.

The case of two structures of the same protein determined by cryo-EM and X-ray crystallography and reported simultaneously is rare. It is reassuring to see that the general features of the two structures coincide, although this was not granted in the particular case of kinesin because the protein it is bound to has different conformations in the two cases. The comparison of the cryo-EM and X-ray results has now proven that crystal structures of kinesin bound to a tubulin heterodimer adequately represent kinesin bound to a microtubule, at least to 6 Å resolution, consistent with the similarity of the biochemical properties of kinesin bound to tubulin or to microtubules.27 But there are local differences, some of which may impact biochemical interpretations. One may need to push the cryo-EM data to a resolution much higher than 6 Å to identify confidently details that differ between the two systems. In what follows, most of the descriptions and discussions will be based on structures determined by X-ray crystallography, in particular because they provide an atomic model that is directly determined.

ATP initiates a mechanical step as it binds to a tubulin-bound kinesin

Each reaction highlighted in Figure 1 is important for the kinesin mechanism, but the one that is the most closely associated to kinesin's mechanochemical properties is the step one motor takes when ATP binds to the other motor domain in a dimer. It has become clear early on that the neck linker, which connects each motor domain to the rest of the molecule, directs the other motor domain toward the microtubule (+)-end as it adopts its docked conformation.9 The question of the mechanochemical mechanism therefore boils down to: what is the link between ATP binding to a nucleotide-free, tubulin-bound, motor domain and neck linker docking?

ATP binding causes local changes in the nucleotide site and, in particular, in the nucleotide binding motifs (Switch1, Switch2, and the P-loop), as expected. But ATP-binding also leads to a general reorganization of the motor domain. This may be described by rigid-body motions of three blocks: a Switch 1/2 subdomain, which contains the Switch1 nucleotide-binding motif and residues of the Switch2 motif, a P-loop subdomain, containing in particular the P-loop [Fig. 2(A)] and a third, tubulin-binding, subdomain at the tubulin-kinesin interface.28,30 ATP, by binding at the interface of the Switch 1/2 and P-loop blocks, favors their reorientation one with respect to the other. Both subdomains also reorient with respect to the tubulin-binding block, which moves little with respect to tubulin. Remarkably, movements of the same subdomains also account for structural changes in the other reactions of the kinesin nucleotide/tubulin binding cycle [Fig. 2(B)].28

Figure 2.

Figure 2

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Kinesin motor subdomains reorient along all the steps of the nucleotide cycle. (A) Nucleotide-free kinesin structure colored by subdomains: Switch 1/2 (cyan), P-loop (beige) and tubulin-binding (green). The nucleotide binding motifs are shown in darker colors (P-loop: dark orange, Switch1: blue and Switch2: dark grey). As the Switch1 loop is disordered in this structure, only part of the Switch1 motif is seen. (B) Subdomains orientation changes along the nucleotide cycle. The rotation axes are schematized taking the microtubule (depicted as a cylinder, with its polarity indicated) as a reference. The neck linker (red) is docked in microtubule-bound kinesin-ATP and undocked in the two other states presented.

As subdomains reorient upon ATP binding, changes in the motor domain occur at a distance from the nucleotide site, and in particular at the other end of the motor domain, 20 Å away from the nucleotide. There, a cavity opens that is boxed in by highly or universally conserved residues in the P-loop and tubulin-binding subdomains. As the neck linker docks on the head, its first residue, a largely conserved isoleucine, gets buried in that cavity (see the enlarged view of this cavity in the rear, ATP-bound, head, with a docked neck linker - Fig. 3). By contrast, in nucleotide-free kinesin, before the movement of the subdomains takes place the cavity is filled, which prevents neck linker docking (see the enlarged view of this region in the front, nucleotide-free, motor domain with an undocked, disordered, neck linker - Fig. 3).

Figure 3.

Figure 3

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Model of the two kinesin motor domains bound to two consecutive tubulin heterodimers in a protofilament, following a step. The model (rear head ATP-like, front head nucleotide-free) was constructed from structures of kinesin bound to tubulin27,28; this represents a transient state that follows front head binding to the microtubule and precedes ATP hydrolysis in the rear head. Enlarged views of the environment of the N-terminal end of the neck linker are shown, framed. Whereas there is space to accommodate the first residue of the neck linker (Ile 325) in the ATP-like structure, the corresponding cavity is closed in the nucleotide-free structure, because of the P-loop subdomain. In nucleotide-free kinesin, the neck linker is disordered.30 Therefore, the conformation of the neck linker presented is only one of the possible ones.

Interestingly, the two conformations of the neck linker (docked and undocked) had been seen in isolated kinesin, much before tubulin–kinesin complexes were available for high-resolution structural studies.12–14 But in that case, as opposed to what happens when kinesin is bound to tubulin, the neck linker seems insensitive to the nucleotide and flips back and forth between the docked and disordered conformations, with either ADP or AMPPNP.9,32 In isolated kinesin, the opening and closure of the cavity where the first residue of the neck linker gets buried is due to a movement of the α4 helix of the tubulin-binding subdomain with respect to the rest of the motor domain, that remains largely unchanged (Fig. 4). By contrast, in tubulin-bound kinesin, neck linker docking is achieved following a large (22°) rotation of the P-loop subdomain. As domain movements are so important for the kinesin mechanism, it is not surprising that some inhibitors work by interfering with these movements. These are the subject of the next section.

Figure 4.

Figure 4

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Superimposed isolated kinesin motor domains structures with docked (pdb id 1MKJ) and undocked (pdb id 1BG2) neck linker (docked: red; undocked: dark orange). The N-terminal end of the undocked neck linker is the only part seen in the structure. Helix α4, which differs most between the two structures, and the neck linker are the only parts of the kinesin with an undocked neck linker presented. In that structure, helix α4 is in dark green.

The mechanism of kinesin inhibitors

As kinesin family proteins play critical roles in many cellular processes, including mitosis, they have been considered as potential targets for anti-cancer drugs and substantial efforts have been invested in the development of inhibitors.33,34 The kinesin spindle protein (known as KSP, or Eg5), a kinesin-5, was the first and is the most studied target. Many of its inhibitors bind to two known allosteric sites away from nucleotide binding site. The first one, in-between helix α2/ Loop L5 and α3, is distant by 10 Å from the nucleotide binding pocket; this site is shared by many extensively characterized drugs, including in particular monastrol (Fig. 5).35 This compound impairs kinesin ATPase by slowing down ADP release.36,37 The second allosteric site of Eg5 is located in-between helices α4 and α6; some of its inhibitors bind tightly to it, with a Kd in the nanomolar range.38 The mechanism of inhibitors at the second site has not been completely clarified yet, except for one compound that causes conformational changes that propagate to the nucleotide-binding site and inhibit ATP binding.39 Interestingly, the two sites are at the interfaces of the P-loop subdomain with the other two kinesin blocks, Switch 1/2 and tubulin-binding for the first and second sites, respectively. As the nucleotide binding site consists of residues of the P-loop and Switch 1/2 subdomains, and as the mechanism of kinesins is largely determined by the movement of the three subdomains, their interfaces seem an ideal binding site for allosteric inhibitors to interfere with kinesin internal reorganization and disturb the nucleotide cycle. Interestingly, some of the inhibitors that bind to the P-loop/Switch 1/2 site favor a docked neck linker conformation in solution,40 a process that mostly depends on the relative orientations of the P-loop and tubulin-binding subdomains.28 Therefore, it seems that interfering with the relative orientations of two motor blocks also affects the third one, illustrating the tight connections between them.

Figure 5.

Figure 5

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Composite view of the sites of the Eg5 kinesin-5 allosteric inhibitors bound to the motor domain. The structure is color-coded according to the motor subdomains as in Figure 2. Inhibitors are shown bound to the two sites identified so far, at the interfaces of the P-loop subdomain with the Switch 1/2 subdomain (monastrol,35 blue) and with the tubulin–binding subdomain (BI8,38 green).

Kinesin is highly processive

Kinesin operates alone or in small numbers: electron micrographs show at most a few linkages between a vesicle and a microtubule.41 Despite that, vesicles move over distances that are much larger than kinesin dimensions, suggesting that each kinesin performs a large number of steps before falling off from a microtubule. The initial demonstration of processivity came from a gliding assay, in which kinesin is attached to a microscope slide and microtubules are watched moving. Experiments done at low kinesin density led to the conclusion that a single kinesin moves a microtubule and accomplishes a large number of steps without detaching.42 A more direct way to watch processivity is a bead assay in which a bead coated with kinesins moves over a microtubule attached to a microscope slide. At low kinesin density, such that there was only one kinesin per bead, beads moved, on average, about 1 µm,16 confirming processivity.

For a dimeric kinesin to move processively, there should always be one motor domain bound to the microtubule. To understand how this happens, a bead assay was used. This showed in particular that the two kinesin motor domains step alternately during movement.6 Further insight was gained as each head was individually tracked using an attached fluorescent label, instead of monitoring the dimer as a whole as in a bead assay. This demonstrated three important properties of kinesin movement, giving rise to the model presented in Figure 6. First, during movement, the kinesin heads are separated by 8 nm most of the time7,43; 8 nm is the length of a tubulin heterodimer, which means that heads are attached to consecutive tubulin molecules on a protofilament. Second, each step corresponds to a 16-nm motion of the rear head toward the microtubule (+)–end, as a result of which it becomes the front head. Finally, forward stepping (the movement of one of the heads upon ATP binding to the other head) is fast (faster than the 500 Hz imaging rate of these experiments43) compared to the dwell time (close to 100 ms43) of the motor domains on microtubules.44,45,43 This reduces the probability of detachment, as the point where a kinesin is prone to detachment during the mechanical cycle is the stage where one head is bound to the microtubule whereas the other one is only tethered to it, as during a step. Taken together, these results support the “hand-over-hand” model for processivity, which proposes that the kinesin stays attached to the microtubule because release of the bound head depends on the binding of the other, so that there is always a head bound.46,5,7

Figure 6.

Figure 6

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One step in the hand-over-hand model of kinesin movement. Upon Pi release, the kinesin returns to its initial conformation, but with exchanged front and rear motor domains.

Because the ATP-bound and nucleotide-free states of the motor domain are the only ones that bind strongly to microtubules,8 there are two prerequisites to processivity. First, the bound head must be kept from hydrolyzing ATP until the ADP-bound tethered head has undergone a 16-nm step. And, second, ADP release by the tethered head must be favored after it has stepped forward, so that it binds strongly to the microtubule while the rear head hydrolyzes ATP, which makes it prone to detachment. Related to this second prerequisite, it was shown that a load externally applied to the neck linker in the direction of the microtubule (–)-end decreases the nucleotide affinity of a microtubule-bound kinesin motor domain.47 As the neck linker of the front, tethered, head is held backward by the bound head, the observations just described explain at the molecular level why nucleotide release by the front head is favored as this head binds to the microtubule. The structural mechanisms for gating of nucleotide release by the neck linker or for ATPase gating, which had remained mysterious,48 have been clarified by the structures recently determined.

Comparison of the structures of tubulin-bound nucleotide-free and ATP-bound kinesin28 shows that, when the neck linker is docked, its first residue is buried in a cavity that is closed in nucleotide-free kinesin (Fig. 3). Therefore, docking of the neck linker prevents the P-loop subdomain from rotating from its ATP-like orientation back to the nucleotide-free one. The relationship between neck linker docking and subdomains orientations has two consequences. First, as neck linker docking locks the structure in an ATP-like structure and, as this is tuned for efficient ATP hydrolysis,27 neck linker docking gates the motor domain ATPase. Consistent with this proposal, a neck linker deletion variant of the motor domain and an Ile -> Gly mutant of the first residue of the neck linker both have a substantially reduced microtubule stimulated ATPase activity, because their P-loop subdomain is not locked anymore in its ATPase competent orientation.28 The interpretation of the structural results and the properties of mutants all lead to conclude that ATP hydrolysis in the bound motor head is not favored until its neck linker is docked, which ensures that stepping occurs. Second, and reciprocally, when the neck linker is held in a direction opposite to motility, as in the front head of a kinesin dimer, it is kept away from its docked position. As a result, the P-loop subdomain of the front head is not locked in its ATP-like structure but it is free to rotate to its nucleotide-free orientation and the nucleotide affinity of the motor domain is expected to be reduced. This is as proposed in kinetic models for kinesin stepping 17 and as suggested by measurements of nucleotide affinity under load.47 In conclusion, the correlation of proper positioning of the three motor subdomains with the conformation of the neck linker is an important contributor to kinesin processivity.

Myosin and kinesin: Relatives, yet different

When the first kinesin structure was determined, a similarity between the kinesin head and the myosin motor domain core was immediately noticed.12 Together with the closely similar topologies of the secondary structure elements of these motor proteins, this led to the proposal that kinesin and myosin derive from a common ancestor, despite their low percentage of sequence identity. Structural studies have recently pointed to two additional features that kinesins have in common with myosins. The myosin motor domain comprises several well-defined subdomains (Fig. 7) and its central β sheet undergoes a twist along its nucleotide cycle49,50 (Fig. 8). By analogy, these two features were postulated for kinesin but have only been observed when structural data on kinesin bound to its track protein became available (Figs. 7 and 8). Most remarkably, the three blocks of the kinesin head correspond closely to three of the main myosin subdomains and the nucleotide binding loops are connected to equivalent subdomains in both proteins. However, and in line with their almost undetectable homology, there are also major differences between the two motor proteins. One of them lies in the manner myosin and kinesin interact with their tracks, actin filaments and microtubules respectively. Whereas the kinesin motor domain binds one tubulin molecule through its tubulin binding subdomain, the myosin head interacts with one actin molecule through elements of the upper 50 (corresponding to the Switch 1/2 kinesin block) and lower 50 (corresponding to the tubulin-binding block) subdomains, and with another actin of the same filament through its lower 50 subdomain.51 The ways myosin and kinesin interact with their respective filaments might be at the origin of two major functional differences, in their nucleotide and mechanical cycles. Whereas in kinesin, upon microtubule binding, both nucleotide exchange (ADP release and subsequent ATP binding) and ATP hydrolysis are enhanced, the actin filament only acts as an exchange factor. Myosin binds to actin in the ADP-Pi state. Upon binding, the inorganic phosphate is released and ADP-ATP exchange takes place, followed by myosin detachment from the filament. In contrast to kinesin, in myosin the chemical step (ATP hydrolysis) takes place in isolated myosin and not when it is bound to the filament. The second major functional difference concerns the mechanical cycle: conformational changes corresponding to force production occur upon Pi release in myosin and upon ATP binding in kinesin. High-resolution data on both motor domains in all their filament-bound states will be required to understand how their interactions with filament proteins lead to such different functional characteristics.

Figure 7.

Figure 7

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Subdomain similarity in myosin and kinesin. (A) Myosin, with bound ADP–BeFx (pdb id 1W7J). (B) Enlarged view in a slightly different orientation of the part framed in (A). (C) Kinesin, with bound ADP–AlFx (pdb id 4HNA). The subdomains are as defined in Cecchini et al.55 for myosin, and in Cao et al.28 for kinesin. The lower 50/tubulin binding subdomains are in green, the N-terminal/P-loop subdomains in orange, and the upper 50/Switch 1/2 subdomains in cyan. Additional myosin subdomains are in pink (converter) or in grey. The P-Loop is in orange, the Switch1 motif in blue, and the Switch2 loop in dark grey for both proteins. The strand just upstream of the P-loop (strand 4 in myosin, strand β3 in kinesin) has been incorporated in the N-terminal subdomain in myosin and in the Switch 1/2 block in kinesin. But in kinesin, this strand, which is at the border between the Switch 1/2 and P-loop blocks, could also have been incorporated in the P-loop block.28

Figure 8.

Figure 8

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Distortion of the central β-sheet upon ATP binding in myosin (upper panel) and in kinesin (lower panel). The structures of the nucleotide-free state (pdb id 1OE9 (myosin) and 4LNU (kinesin)) and of an ATP-like state (pdb id 1W7J (myosin) and 4HNA (kinesin)) have been superimposed on strands that belong to the N-terminal/P-loop subdomains (strands 1 to 4 for myosin, and β1, β2 and β8 for kinesin; these strands are displayed only in the ATP-like structures). Strands of the upper 50/Switch 1/2 subdomains are in cyan (ATP-like structures) or in blue (nucleotide-free structures).

Perspectives

The recent developments of kinesin structural studies provide the proper framework to rationally design mutants that clarify aspects of the biochemical mechanism of this motor protein, such as the gating of microtubule-stimulated ATPase by the neck linker.28 The new structures have also been used to explain the properties of previously characterized mutants.30 Other structure-function studies have only been started. This is in particular the case of tight ADP binding by the isolated kinesin and of microtubule-stimulated ADP release. The property is fundamental to the efficiency of kinesin as it prevents futile ATP hydrolysis when the motor is not bound to a microtubule, that is, is not transporting any load. Mutants were designed that accelerate ADP release by the isolated kinesin substantially28 but the microtubule stimulated rate has not been reached yet. Hopefully, further work will soon complete our understanding of this important property.

Two reactions in the nucleotide cycle are still poorly understood. We do not have a structural mechanism for phosphate release and the interaction of ADP-kinesin with microtubules has not been satisfactorily explained either. When an ADP-kinesin dimer initially binds to a microtubule, the ADP in the bound head is released, giving rise to a nucleotide-free kinesin motor tightly bound to microtubule. But when ATP is hydrolyzed in the microtubule-bound rear head of a dimer, which also produces a kinesin-ADP bound to microtubule, this leads to detachment of this motor domain.52 A tentative explanation of the first part of this dual behavior is that when ADP-kinesin initially binds to a microtubule, the neck linker is undocked so that nucleotide release is facilitated. But the mechanism that leads to rear head detachment still needs to be established and would warrant further studies.

Although the general features of conventional kinesin mechanism are becoming clear, much remains to be done, even only concerning other (+)-end directed motile kinesins. Indeed, the sequences and properties of these proteins differ substantially. Some of them move faster than conventional kinesin,53 whereas the movement of others is extremely slow and inefficient, as several ATP molecules get hydrolyzed per step.54 Deciphering how the different properties are related to these kinesins’ sequences will require in particular much further structural analysis. Because methods are now available to obtain high-resolution structural data on most of the states of the mechanochemical cycle of kinesins, this goal seems within reach.

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