Configuration of the two kinesin motor domains during ATP hydrolysis

To understand the mechanism of kinesin movement we have investigated the relative configuration of the two kinesin motor domains during ATP hydrolysis using fluorescence polarization microscopy of ensemble and single molecules. We found that: (i) in nucleotide states that induce strong microtubule binding, both motor domains are bound to the microtubule with similar orientations; (ii) this orientation is maintained during processive motion in the presence of ATP; (iii) the neck-linker region of the motor domain has distinct configurations for each nucleotide condition tested. Our results fit well with a hand-over-hand type movement mechanism and suggest how the ATPase cycle in the two motor domains is coordinated. We propose that the motor neck-linker domain configuration controls ADP release.     

Crystal structure of the mitotic spindle kinesin Eg5 reveals a novel conformation of the neck-linker

Success of mitosis depends upon the coordinated and regulated activity of many cellular factors, including kinesin motor proteins, which are required for the assembly and function of the mitotic spindle. Eg5 is a kinesin implicated in the formation of the bipolar spindle and its movement prior to and during anaphase. We have determined the crystal structure of the Eg5 motor domain with ADP-Mg bound. This structure revealed a new intramolecular binding site of the neck-linker. In other kinesins, the neck-linker has been shown to be a critical mechanical element for force generation. The neck-linker of conventional kinesin is believed to undergo an ordered-to-disordered transition as it translocates along a microtubule. The structure of Eg5 showed an ordered neck-linker conformation in a position never observed previously. The docking of the neck-linker relies upon residues conserved only in the Eg5 subfamily of kinesin motors. Based on this new information, we suggest that the neck-linker of Eg5 may undergo an ordered-to-ordered transition during force production. This ratchet-like mechanism is consistent with the biological activity of Eg5.     

Kinesin’s Neck-Linker Determines its Ability to Navigate Obstacles on the Microtubule Surface

The neck-linker is a structurally conserved region among most members of the kinesin superfamily of molecular motor proteins that is critical for kinesin’s processive transport of intracellular cargo along the microtubule surface. Variation in the neck-linker length has been shown to directly modulate processivity in different kinesin families; for example, kinesin-1, with a shorter neck-linker, is more processive than kinesin-2. Although small differences in processivity are likely obscured in vivo by the coupling of most cargo to multiple motors, longer and more flexible neck-linkers may allow different kinesins to navigate more efficiently around the many obstacles, including microtubule-associated proteins (MAPs), that are found on the microtubule surface within cells. We hypothesize that, due to its longer neck-linker, kinesin-2 can more easily navigate obstacles (e.g., MAPs) on the microtubule surface than kinesin-1. We used total internal reflection fluorescence microscopy to observe single-molecule motility from different kinesin-1 and kinesin-2 neck-linker chimeras stepping along microtubules in the absence or presence of two Tau isoforms, 3RS-Tau and 4RL-Tau, both of which are MAPs that are known to differentially affect kinesin-1 motility. Our results demonstrate that unlike kinesin-1, kinesin-2 is insensitive to the presence of either Tau isoform, and appears to have the ability to switch protofilaments while stepping along the microtubule when challenged by an obstacle, such as Tau. Thus, although kinesin-1 may be more processive, the longer neck-linker length of kinesin-2 allows it to be better optimized to navigate the complex microtubule landscape. These results provide new insight, to our knowledge, into how kinesin-1 and kinesin-2 may work together for the efficient delivery of cargo in cells.     

Kinesin: a molecular motor with a spring in its step

A key step in the processive motion of two-headed kinesin along a microtubule is the 'docking' of the neck linker that joins each kinesin head to the motor's dimerized coiled-coil neck. This process is similar to the folding of a protein beta-hairpin, which starts in a highly mobile unfolded state that has significant entropic elasticity and finishes in a more rigid folded state. We therefore suggest that neck-linker docking is mechanically equivalent to the thermally activated shortening of a spring that has been stretched by an applied load. This critical tension-dependent step utilizes Brownian motion and it immediately follows the binding of ATP, the hydrolysis of which provides the free energy that drives the kinesin cycle. A simple three-state model incorporating neck-linker docking can account quantitatively for both the kinesin force-velocity relation and the unusual tension-dependence of its Michaelis constant. However, we find that the observed randomness of the kinesin motor requires a more detailed four-state model. Monte Carlo simulations of single-molecule stepping with this model illustrate the possibility of sub-8 nm steps, the size of which is predicted to vary linearly with the applied load.     

Mapping the Structural and Dynamical Features of Kinesin Motor Domains

Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes that mediate directed movement along microtubules. Characterizing these distinct conformations and their interconversion mechanism is essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of conventional and accelerated molecular dynamics simulations that together map the structural dynamics of the kinesin motor domain. All experimental structures were found to reside in one of three distinct conformational clusters (ATP-like, ADP-like and Eg5 inhibitor-bound). These groups differ in the orientation of key functional elements, most notably the microtubule binding α4–α5, loop8 subdomain and α2b-β4-β6-β7 motor domain tip. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. However, groupings were coincident with distinct neck-linker orientations. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicate that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were also evident. In multiple ATP bound simulations, the neck-linker, loop8 and the α4–α5 subdomain display correlated motions that are absent in ADP bound simulations. Further dissection of these couplings provides evidence for a network of dynamic communication between the active site, microtubule-binding interface and neck-linker via loop7 and loop13. Additional simulations indicate that the mutations G325A and G326A in loop13 reduce the flexibility of these regions and disrupt their couplings. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state and support a model where neck-linker docking leads to a tighter coupling of the microtubule and nucleotide binding regions. Furthermore, simulations highlight sites critical for large-scale conformational changes and the allosteric coupling between distal functional sites.     

Nucleotide binding and hydrolysis induces a disorder-order transition in the kinesin neck-linker region

Kinesin translocation is thought to occur by a conformational change in a region of the motor domain called the neck linker. However, most evidence supporting this hypothesis comes from monomeric constructs unable to move processively. To address this issue, we investigated the neck-linker configuration on microtubule-bound monomeric and dimeric kinesin constructs using single-molecule fluorescence polarization microscopy. We found that the neck-linker region (i) is very mobile in the absence of nucleotides and during steady walking, (ii) decreases mobility and aligns along the microtubule axis in the presence of AMPPNP or ADP + AlF4(-), (iii) is mostly ordered in the monomeric constructs in the presence of ADP + AlF4(-), and (iv) is closer to parallel to the microtubule axis in the dimeric constructs. These results support the proposed role of the neck linker and suggest a coordination mechanism between the two motor domains in the dimer.     

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.     

Kinesin, 30 years later: Recent insights from structural studies

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.     

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.     

Kinesin-5 Allosteric Inhibitors Uncouple the Dynamics of Nucleotide,Microtubule, and Neck-Linker Binding Sites

Kinesin motor domains couple cycles of ATP hydrolysis to cycles of microtubule binding and conformational changes that result in directional force and movement on microtubules. The general principles of this mechanochemical coupling have been established; however, fundamental atomistic details of the underlying allosteric mechanisms remain unknown. This lack of knowledge hampers the development of new inhibitors and limits our understanding of how disease-associated mutations in distal sites can interfere with the fidelity of motor domain function. Here, we combine unbiased molecular-dynamics simulations, bioinformatics analysis, and mutational studies to elucidate the structural dynamic effects of nucleotide turnover and allosteric inhibition of the kinesin-5 motor. Multiple replica simulations of ATP-, ADP-, and inhibitor-bound states together with network analysis of correlated motions were used to create a dynamic protein structure network depicting the internal dynamic coordination of functional regions in each state. This analysis revealed the intervening residues involved in the dynamic coupling of nucleotide, microtubule, neck-linker, and inhibitor binding sites. The regions identified include the nucleotide binding switch regions, loop 5, loop 7, α4-α5-loop 13, α1, and β4-β6-β7. Also evident were nucleotide- and inhibitor-dependent shifts in the dynamic coupling paths linking functional sites. In particular, inhibitor binding to the loop 5 region affected β-sheet residues and α1, leading to a dynamic decoupling of nucleotide, microtubule, and neck-linker binding sites. Additional analyses of point mutations, including P131 (loop 5), Q78/I79 (α1), E166 (loop 7), and K272/I273 (β7) G325/G326 (loop 13), support their predicted role in mediating the dynamic coupling of distal functional surfaces. Collectively, our results and approach, which we make freely available to the community, provide a framework for explaining how binding events and point mutations can alter dynamic couplings that are critical for kinesin motor domain function.     

Two conformations in the human kinesin power stroke defined by X-ray crystallography and EPR spectroscopy

Crystal structures of the molecular motor kinesin show conformational variability in a structural element called the neck linker. Conformational change in the neck linker, initiated by ATP exchange, is thought to drive the movement of kinesin along the microtubule track. We use site-specific EPR measurements to show that when microtubules are absent, the neck linker exists in equilibrium between two structural states (disordered and 'docked'). The active site nucleotide does not control the position taken by the neck linker. However, we find that sulfate can specifically bind near the nucleotide site and stabilize the docked neck linker conformation, which we confirmed by solving a new crystal structure. Comparing the crystal structures of our construct with the docked or undocked neck linker reveals how microtubule binding may activate the nucleotide-sensing mechanism of kinesin, allowing neck linker transitions to power motility.     

Kinesin-5 Allosteric Inhibitors Uncouple the Dynamics of Nucleotide,Microtubule, and Neck-Linker Binding Sites

Kinesin motor domains couple cycles of ATP hydrolysis to cycles of microtubule binding and conformational changes that result in directional force and movement on microtubules. The general principles of this mechanochemical coupling have been established; however, fundamental atomistic details of the underlying allosteric mechanisms remain unknown. This lack of knowledge hampers the development of new inhibitors and limits our understanding of how disease-associated mutations in distal sites can interfere with the fidelity of motor domain function. Here, we combine unbiased molecular-dynamics simulations, bioinformatics analysis, and mutational studies to elucidate the structural dynamic effects of nucleotide turnover and allosteric inhibition of the kinesin-5 motor. Multiple replica simulations of ATP-, ADP-, and inhibitor-bound states together with network analysis of correlated motions were used to create a dynamic protein structure network depicting the internal dynamic coordination of functional regions in each state. This analysis revealed the intervening residues involved in the dynamic coupling of nucleotide, microtubule, neck-linker, and inhibitor binding sites. The regions identified include the nucleotide binding switch regions, loop 5, loop 7, α4-α5-loop 13, α1, and β4-β6-β7. Also evident were nucleotide- and inhibitor-dependent shifts in the dynamic coupling paths linking functional sites. In particular, inhibitor binding to the loop 5 region affected β-sheet residues and α1, leading to a dynamic decoupling of nucleotide, microtubule, and neck-linker binding sites. Additional analyses of point mutations, including P131 (loop 5), Q78/I79 (α1), E166 (loop 7), and K272/I273 (β7) G325/G326 (loop 13), support their predicted role in mediating the dynamic coupling of distal functional surfaces. Collectively, our results and approach, which we make freely available to the community, provide a framework for explaining how binding events and point mutations can alter dynamic couplings that are critical for kinesin motor domain function.     

Helix capping interactions stabilize the N-terminus of the kinesin neck coiled-coil

In an effort to understand how specific structural features within the kinesin neck, a region of the heavy chain located between the catalytic core and stalk domains, may contribute to motor processivity (an ability to remain attached to the microtubule filament), we have prepared several synthetic peptides corresponding to the neck region of human conventional kinesin and determined their secondary structure content and stability by CD spectroscopy. Our results show that the coiled-coil dimerization domain within the human kinesin neck region corresponds to residues 337 to 369 in solution, and thus is in excellent agreement with the recent X-ray crystallographic structures of rat brain kinesin. Further, we show that the first and last heptads of this region are absolutely critical for creating the high stability and association of the dimeric structure. Interestingly, addition of the 7 N-terminal neck-linker residues (330-336) to the coiled-coil domain significantly increased its stability (Delta GdnHCl midpoint of 1 M or an increase of approximately 1.5 kcal/mol), indicating that a strong structural link exists between the neck-linker and coiled-coil region. Subsequent high-resolution structural analysis of the residues located at the junction of the neck-linker and coiled-coil revealed the presence of the two helix capping motifs, the capping box (a reciprocal interaction of Thr 336 with Gln 339) and the hydrophobic staple (a hydrophobic packing interaction of Leu 335 with Trp 340). Substitution of Leu 335 and Thr 336 (the capping residues) with Gly completely eliminated the increased stability of the coiled-coil region observed in the presence of the neck-linker residues. Correspondingly, substitution of Trp 340, the first hydrophobic core d position residue of the coiled-coil, with an Ala residue resulted in a greater than expected decrease in stability and helicity of the coiled-coil structure. Subsequent analysis of the X-ray structure and substitution analysis of Lys 341 revealed that Trp 340 makes an important interchain hydrophobic interaction with Lys 341 of the opposite chain. Taken together these results reveal that a set of strong intra- and inter-chain interactions made up of the helix "capping box," "hydrophobic staple," and the newly identified "Leu-Trp-Lys sandwich" motifs stabilize the kinesin neck coiled-coil structure, thus preventing it from fraying and unfolding.     

Role of the kinesin neck linker and catalytic core in microtubule-based motility

Kinesin motor proteins execute a variety of intracellular microtubule-based transport functions [1]. Kinesin motor domains contain a catalytic core, which is conserved throughout the kinesin superfamily, followed by a neck region, which is conserved within subfamilies and has been implicated in controlling the direction of motion along a microtubule [2] [3]. Here, we have used mutational analysis to determine the functions of the catalytic core and the approximately 15 amino acid 'neck linker' (a sequence contained within the neck region) of human conventional kinesin. Replacement of the neck linker with a designed random coil resulted in a 200-500-fold decrease in microtubule velocity, although basal and microtubule-stimulated ATPase rates were within threefold of wild-type levels. The catalytic core of kinesin, without any additional kinesin sequence, displayed microtubule-stimulated ATPase activity, nucleotide-dependent microtubule binding, and very slow plus-end-directed motor activity. On the basis of these results, we propose that the catalytic core is sufficient for allosteric regulation of microtubule binding and ATPase activity and that the kinesin neck linker functions as a mechanical amplifier for motion. Given that the neck linker undergoes a nucleotide-dependent conformational change [4], this region might act in an analogous fashion to the myosin converter, which amplifies small conformational changes in the myosin catalytic core [5,6].     

A mechanistic model for Ncd directionality

Ncd is a kinesin-related protein that drives movement to the minus-end of microtubules. Pre-steady-state kinetic experiments have been employed to investigate the cooperative interactions between the motor domains of the MC1 dimer and to establish the ATPase mechanism. Our results indicate that the active sites of dimeric Ncd free in solution are not equivalent; ADP is held more tightly at one site than at the other. Upon microtubule binding, fast release of ADP from the first motor domain is stimulated at 18 s(-1), yet rate-limiting ADP release from the second motor domain occurs at 1.4 s(-1). We propose that the head with the low affinity for ADP binds the microtubule first to establish the directional bias of the microtubule.Ncd intermediate where one motor domain is bound to the microtubule with the second head detached and directed toward the minus-end of the microtubule. The force generating cycle is initiated as ATP binds to the empty site of the microtubule-bound head. ATP hydrolysis at head 1 is required for head 2 to bind to the microtubule. The kinetics indicate that two ATP molecules are required for a single step and force generation for minus-end directed movement generated by this non-processive dimeric motor.     

Controlling kinesin motor proteins in nanoengineered systems through a metal-binding on/off switch

A significant challenge in utilizing kinesin biomolecular motors in integrated nanoscale systems is the ability to regulate motor function in vitro. Here we report a versatile mechanism for reversibly controlling the function of kinesin biomolecular motors independent of the fuel supply (ATP). Our approach relied on inhibiting conformational changes in the neck-linker region of kinesin, a process necessary for microtubule transport. We introduced a chemical switch into the neck-linker of kinesin by genetically engineering three histidine residues to create a Zn(2+)-binding site. Gliding motility of microtubules by the mutant kinesin was successfully inhibited by >/=10 microM Zn(2+), as well as other divalent metals. Motility was successfully restored by removal of Zn(2+) using a number of different chelators. Lastly, we demonstrated the robust and cyclic nature of the switch using sequential Zn(2+)/chelator additions. Overall, this approach to controlling motor function is highly advantageous as it enables control of individual classes of biomolecular motors while maintaining a consistent level of fuel for all motors in a given system or device.     

Origins of reversed directionality in the ncd molecular motor.

The head or motor domain of the ncd (non-claret disjunctional) molecular motor is 41% identical to that of kinesin, yet moves along microtubules in the opposite direction to kinesin. We show here that despite the reversed directionality of ncd, its kinetics in solution are homologous in key respects to those of kinesin. The rate limiting step, ADP release, occurs at 0.0033 s-1 at 100 mM NaCl and is accelerated approximately 1000-fold when the motor binds to microtubules. Other reaction steps are all very fast (> 0.1 s-1) compared with ADP release, and the motor is consequently paused in the ncd.ADP state until microtubule binding occurs (Kd = 2 microM), at which point ADP release is triggered and the motor locks onto the microtubule in a rigor-like state. These data identify close functional homology between the strong binding states of kinesin and ncd, and in view of this we discuss a possible mechanism for directional reversal, in which the strong binding states of ncd and kinesin are functionally identical, but the weak binding states are biased in opposite directions.     

Mechanism of tail-mediated inhibition of kinesin activities studied using synthetic peptides

We used a truncated form of human conventional kinesin (K560) and a set of synthetic tail-derived peptides to investigate the mechanism by which the kinesin tail domain inhibits the protein's ATPase and motor activities. A peptide that spans residues 904-933 (C3) exhibited the strongest inhibitory effect on steady-state motility and ATPase activity. This inhibition reflected diminished binding of the ADP-bound kinesin head to the microtubule. Although peptide C3 bound to both K560 and microtubules, gliding assays using subtilisin-treated microtubules suggested that the binding to the microtubule contributes only little to the inhibition if there is sufficient affinity between the peptide and kinesin. We suggest that tail-mediated inhibition of kinesin activity is mainly the product of allosteric inhibition induced by the intramolecular binding of the kinesin tail domain to the motor domain, but simultaneous binding of the tail to the microtubule also may exert a minor effect.     

Hopping and stalling of processive molecular motors

When a two-headed molecular motor such as kinesin is attached to its track by just a single head in the presence of an applied load, thermally activated head detachment followed by rapid re-attachment at another binding site can cause the motor to ‘hop’ backwards. Such hopping, on its own, would produce a linear force-velocity relation. However, for kinesin, we must incorporate hopping into the motor's alternating-head scheme, where we expect it to be most important for the state prior to neck-linker docking. We show that hopping can account for the backward steps, run length and stalling of conventional kinesin. In particular, although hopping does not hydrolyse ATP, we find that the hopping rate obeys the same Michaelis-Menten relation as the ATP hydrolysis rate. Hopping can also account for the reduced processivity observed in kinesins with mutations in their tubulin-binding loop. Indeed, it may provide a general mechanism for the breakdown of perfect processivity in two-headed molecular motors.     

ESR reveals the mobility of the neck linker in dimeric kinesin

Conventional kinesin is a highly processive motor that converts the chemical energy of ATP hydrolysis into the unidirectional motility along microtubules. The processivity is thought to depend on the coordination between ATPase cycles of two motor domains and their neck linkers. Here we have used site-directed spin labeling electron spin resonance (SDSL-ESR) to determine the conformation of the neck linker in kinesin dimer in the presence and absence of microtubules. The spectra show that the neck linkers co-exist in both docked and disordered conformations, which is consistent with the results of monomeric kinesin. In all nucleotide states, however, the neck linkers are well ordered when dimeric kinesin is bound to the microtubule. This result suggests that the orientation of each neck linker that is fixed rigidly controls the kinesin motion along microtubule tracks.