3D electron microscopy of the interaction of kinesin with tubulin

We have studied the structure of microtubules decorated with kinesin motor domains in different nucleotide states by 3D electron microscopy. Having docked the atomic coordinates of both dimeric ADP.kinesin and tubulin heterodimer into a map of kinesin dimers bound to microtubules in the presence of ADP, we try to predict which regions of the proteins interact in the weakly binding state. When either the presence of 5'-adenylyimidodiphosphate (AMP-PNP) or an absence of nucleotides puts motor domains into a strongly-bound state, the 3D maps show changes in the motor domains which modify their interaction with beta-tubulin. The maps also show differences in beta-tubulin conformation compared with undecorated microtubules or those decorated with weakly-bound motors. Strongly-bound ncd appears to produce an identical change.     

Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules

Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.     

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.     

X‐ray and Cryo‐EM structures reveal mutual conformational changes of Kinesin and GTP‐state microtubules upon binding

The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin‐1/KIF5C preferentially binds to the GTP‐state microtubules over GDP‐state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide‐free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide‐free KIF5C and the cryo‐electron microscopic structure of nucleotide‐free KIF5C complexed with the GTP‐state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP‐state microtubule and KIF5C. KIF5C acquires the ‘rigor conformation’, where mobile switches I and II are stabilized through L11 and the initial portion of the neck‐linker, facilitating effective ADP release and the weak‐to‐strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP‐state microtubule in a similar manner to GDP‐taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP‐state microtubules.     

The structure of the nucleotide-binding site of kinesin.

Kinesin is a microtubule-based motor protein responsible for anterograde transport of vesicles and organelles in nerve axons and other cell types. The energy necessary for this transport is derived from the hydrolysis of ATP which is thought to induce conformational changes in the protein. We have solved the X-ray crystal structures of rat brain kinesin in three conditions intended to mimic different nucleotide states: (1) with ADP bound to the nucleotide-binding site, (2) with bound ADP in the presence of AIF(-)4, and (3) with ADP hydrolyzed to AMP by apyrase. In contrast to analogous cases observed in GTP-binding proteins or the muscle motor myosin, the structure of kinesin remained nearly unchanged. This highlights the stability of kinesin's ADP state in the absence of microtubules. Surprisingly, even after hydrolysis of ADP to AMP by apyrase a strong density peak remains at the position of the beta-phosphate which is compatible either with a phosphate or a sulfate from the solvent and appears to stabilize the nucleotide-binding pocket through several hydrogen bonds.     

Diffusive Movement of Processive Kinesin-1 on Microtubules

The processive motor kinesin-1 moves unidirectionally toward the plus end of microtubules. This process can be visualized by total internal reflection fluorescence microscopy of kinesin bound to a carboxylated quantum dot (Qdot), which acts both as cargo and label. Surprisingly, when kinesin is bound to an anti-HIS Qdot, it shows diffusive movement on microtubules, which decreased in favor of processive runs with increasing salt concentration. This observation implies that kinesin movement on microtubules is governed by its conformation, as it is well established that kinesin undergoes a salt-dependent transition from a folded (inactive) to an extended (active) molecule. A truncated kinesin lacking the last 75 amino acids (kinesin-ΔC) showed both processive and diffusive movement on microtubules. The extent of each behavior depends on the relative amounts of ADP and ATP, with purely diffusive movement occurring in ADP alone. Taken together, these data imply that folded kinesin.ADP can exist in a state that diffuses along the microtubule lattice without expending energy. This mechanism may facilitate the ability of kinesin to pick up cargo, and/or allow the kinesin/cargo complex to stay bound after encountering obstacles.     

EPR spectroscopy shows a microtubule-dependent conformational change in the kinesin switch 1 domain

We have used site-directed spin-labeling and electron paramagnetic resonance spectroscopy to monitor a conformational change at the nucleotide site of kinesin. Cys-lite kinesin (K349 monomer) with the mutation S188C was spin labeled with MSL or MTSL. This residue is at the junction between the switch 1 region (which is a structure known to be sensitive to bound nucleotide in the G-proteins) and the alpha3-helix, adjacent to the nucleotide site. The spectra showed two or more components of mobility, which were independent of nucleotide in the absence of microtubules (MTs). The spectra of both labels showed a change of mobility upon binding to MTs. A more mobile spectral component became enhanced for all triphosphate analogs examined, AMPPNP, ADP.AlFx, or ADP.BeFx, in the presence of MTs, although the magnitude of the new component and the degree of mobility varied with nucleotide analog. The ADP state showed a much-reduced spectral change with a small shift to the more immobilized component in the presence of MTs. For kinesin.ADP.MT, a van't Hoff plot gave DeltaH degrees = -96 kJ/mol implying that the conformational change was extensive. We conclude there is a conformational change in the switch 1-alpha3-helix domain when kinesin binds to MTs.     

The kinesin-13 MCAK has an unconventional ATPase cycle adapted for microtubule depolymerization

Unlike other kinesins, members of the kinesin-13 subfamily do not move directionally along microtubules but, instead, depolymerize them. To understand how kinesins with structurally similar motor domains can have such dissimilar functions, we elucidated the ATP turnover cycle of the kinesin-13, MCAK. In contrast to translocating kinesins, ATP cleavage, rather than product release, is the rate-limiting step for ATP turnover by MCAK; unpolymerized tubulin and microtubules accelerate this step. Further, microtubule ends fully activate the ATPase by accelerating the exchange of ADP for ATP. This tuning of the cycle adapts MCAK for its depolymerization activity: lattice-stimulated ATP cleavage drives MCAK into a weakly bound nucleotide state that reaches microtubule ends by diffusion, and end-specific acceleration of nucleotide exchange drives MCAK into a strongly bound state that promotes depolymerization. This altered cycle accounts well for the different mechanical behaviour of this kinesin, which depolymerizes microtubules from their ends, compared to translocating kinesins that walk along microtubules. Thus, the kinesin motor domain is a nucleotide-dependent engine that can be differentially tuned for transport or depolymerization functions.     

Congruent Docking of Dimeric Kinesin and ncd into Three-dimensional Electron Cryomicroscopy Maps of Microtubule–Motor ADP Complexes

We present a new map showing dimeric kinesin bound to microtubules in the presence of ADP that was obtained by electron cryomicroscopy and image reconstruction. The directly bound monomer (first head) shows a different conformation from one in the more tightly bound empty state. This change in the first head is amplified as a movement of the second (tethered) head, which tilts upward. The atomic coordinates of kinesin.ADP dock into our map so that the tethered head associates with the bound head as in the kinesin dimer structure seen by x-ray crystallography. The new docking orientation avoids problems associated with previous predictions; it puts residues implicated by proteolysis-protection and mutagenesis studies near the microtubule but does not lead to steric interference between the coiled-coil tail and the microtubule surface. The observed conformational changes in the tightly bound states would probably bring some important residues closer to tubulin. As expected from the homology with kinesin, the atomic coordinates of nonclaret disjunctional protein (ncd).ADP dock in the same orientation into the attached head in a map of microtubules decorated with dimeric ncd.ADP. Our results support the idea that the observed direct interaction between the two heads is important at some stages of the mechanism by which kinesin moves processively along microtubules.     

X-ray structure and microtubule interaction of the motor domain of Neurospora crassa NcKin3, a kinesin with unusual processivity

Neurospora crassa kinesin NcKin3 belongs to a unique fungal-specific subgroup of small Kinesin-3-related motor proteins. One of its functions appears to be the transport of mitochondria along microtubules. Here, we present the X-ray structure of a C-terminally truncated monomeric construct of NcKin3 comprising the motor domain and the neck linker, and a 3-D image reconstruction of this motor domain bound to microtubules, by cryoelectron microscopy. The protein contains Mg.ADP bound to the active site, yet the structure resembles an ATP-bound state. By comparison with structures of the Kinesin-3 motor Kif1A in different nucleotide states (Kikkawa, M. et al. (2001) Nature (London, U.K.) 411, 439-445), the NcKin3 structure corresponds to the AMPPCP complex of Kif1A rather than the AMPPNP complex. NcKin3-specific differences in the coordination of the nucleotide and asymmetric interactions between adjacent molecules in the crystal are discussed in the context of the unusual kinetics of the dimeric wild-type motor and the monomeric construct used for crystal structure analysis. The NcKin3 motor decorates microtubules at a stoichiometry of one head per alphabeta-tubulin heterodimer, thereby forming an axial periodicity of 8 nm. In spite of unusual extensions at the N-terminus and within flexible loops L2, L8a, and L12 (corresponding to the K-loop of monomeric kinesins), the microtubule binding geometry is similar to that of other members of the kinesin family.     

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.     

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.     

Large conformational changes in a kinesin motor catalyzed by interaction with microtubules

Kinesin motor proteins release nucleotide upon interaction with microtubules (MTs), then bind and hydrolyze ATP to move along the MT. Although crystal structures of kinesin motors bound to nucleotides have been solved, nucleotide-free structures have not. Here, using cryomicroscopy and three-dimensional (3D) reconstruction, we report the structure of MTs decorated with a Kinesin-14 motor, Kar3, in the nucleotide-free state, as well as with ADP and AMPPNP, with resolution sufficient to show alpha helices. We find large structural changes in the empty motor, including melting of the switch II helix alpha4, closure of the nucleotide binding pocket, and changes in the central beta sheet reminiscent of those reported for nucleotide-free myosin crystal structures. We propose that the switch II region of the motor controls docking of the Kar3 neck by conformational changes in the central beta sheet, similar to myosin, rather than by rotation of the motor domain, as proposed for the Kif1A kinesin motor.     

High-resolution cryo-EM maps show the nucleotide binding pocket of KIF1A in open and closed conformations

Kinesin is an ATP-driven microtubule (MT)-based motor fundamental to organelle transport. Although a number of kinesin crystal structures have been solved, the structural evidence for coupling between the bound nucleotide and the conformation of kinesin is elusive. In addition, the structural basis of the MT-induced ATPase activity of kinesin is not clear because of the absence of the MT in the structure. Here, we report cryo-electron microscopy structures of the monomeric kinesin KIF1A-MT complex in two nucleotide states at about 10 A resolution, sufficient to reveal the secondary structure. These high-resolution maps visualized clear structural changes that suggest a mechanical pathway from the nucleotide to the neck linker via the motor core rotation. In addition, new nucleotide binding pocket conformations are observed that are different from X-ray crystallographic structures; it is closed in the 5'-adenylyl-imidodiphosphate state, but open in the ADP state. These results suggest a structural model of biased diffusion movement of monomeric kinesin motor.     

Removal of tightly bound ADP induces distinct structural changes of the two tryptophan-containing regions of the ncd motor domain

ncd is a molecular motor belonging to the kinesin superfamily. In solution, it is a homo-dimer of a 700 amino acid polypeptide. The C-terminus of each polypeptide forms a globular domain of about 40 kDa, the motor domain with ATPase activity. The ATPase site of the motor domain of kinesin family members, including ncd, binds ADP tightly, the release of which is facilitated by microtubules during the mechanochemical ATPase cycle. Previously, we studied the spectroscopic characteristics of the ncd motor domain, focusing on interactions of the transition-moment-dipoles between ADP and aromatic amino acid side chains using circular dichroism (CD) spectroscopy. In the present study, we generated several ncd motor domain mutants. In each, a tryptophanyl or specific tyrosyl residue was mutated. We found that Trp370 and Tyr442, the latter of which stacks directly with the adenine moiety of bound ADP, caused the bound ADP to exhibit peculiar CD signals. In addition, fluorescence measurements revealed that Trp370, but not Trp473, was responsible for the emission intensity change depending on the presence or absence of bound ADP. This fluorescence result implies that the structural change induced at the ADP-binding site (on the release of the ADP) is transmitted to the region that includes Trp370, which is relatively close to the ADP-binding site but not in direct contact with the ADP-binding region. In contrast, Trp473 in the region that is in contact with the alpha-helical coiled coil stalk did not experience the structural changes caused on removal of ADP. The distinct behavior of these two tryptophanyl residues suggests that the ncd motor domain has a bifacial architecture made up of a relatively deformable side including the nucleotide binding site and a more rigid one.     

Nucleotide-dependent displacement and dynamics of the α-1 helix in kinesin revealed by site-directed spin labeling EPR

In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening. The inter-helix distance distribution showed that 20% of the spins have a peak characteristic of 1.4–1.7 nm separation, which is similar to what is predicted from the X-ray crystal structure, albeit 80% were beyond the sensitivity limit (>2.5 nm) of the method. Upon MT binding, the fraction of kinesin exhibiting an inter-helix distance of 1.4–1.7 nm in the presence of AMPPNP (a non-hydrolysable ATP analog) and ADP was 20% and 25%, respectively. In the absence of nucleotide, this fraction increased to 40–50%. These nucleotide-induced changes in the fraction of kinesin undergoing displacement of the α-1 helix were found to be related to the fraction in which the NL undocked from the motor core. It is therefore suggested that a shift in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.     

Formation and characterization of kinesin.ADP.fluorometal complexes

Recent crystallographic studies of motor proteins showed that the structure of the motor domains of myosin and kinesin are highly conserved. Thus, these motor proteins, which are important for motility, may share a common mechanism for generating energy from ATP hydrolysis. We have previously demonstrated that, in the presence of ADP, myosin forms stable ternary complexes with new phosphate analogues of aluminum fluoride (AlF(4)(-)) and beryllium fluoride (BeF(n)), and these stable complexes mimic the transient state along the ATPase kinetic pathway [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. In this study, we examined the formation of kinesin.ADP.fluorometals ternary complexes and analyzed their characteristics using the fluorescent ATP analogue NBD-ATP (2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl]-ADP). Our results suggest that these ternary complexes may mimic transient state intermediates in the kinesin ATPase cycle. Thus, the kinesin.ADP.AlF(4)(-) complex resembles the kinesin.ADP state, and the kinesin.ADP.BeF(n) complex mimics the kinesin.ADP.P(i) state.     

Multiple conformations of the nucleotide site of Kinesin family motors in the triphosphate state

Identifying conformational changes in kinesin family motors associated with nucleotide and microtubule (MT) binding is essential to determining an atomic-level model for force production and motion by the motors. Using the mobility of nucleotide analog spin probes bound at the active sites of kinesin family motors to monitor conformational changes, we previously demonstrated that, in the ADP state, the open nucleotide site closes upon MT binding [Naber, N., Minehardt, T. J., Rice, S., Chen, X., Grammer, J., Matuska, M., et al. (2003). Closing of the nucleotide pocket of kinesin family motors upon binding to microtubules. Science, 300, 798-801]. We now extend these studies to kinesin-1 (K) and ncd (nonclaret disjunctional protein) motors in ATP and ATP-analog states. Our results reveal structural differences between several triphosphate and transition-state analogs bound to both kinesin and ncd in solution. The spectra of kinesin/ncd in the presence of SLADP•AlFx/BeFx and kinesin, with the mutation E236A (K-E236A; does not hydrolyze ATP) bound to ATP, show an open conformation of the nucleotide pocket similar to that seen in the kinesin/ncd•ADP states. In contrast, the triphosphate analogs K•SLAMPPNP and K-E236A•SLAMPPNP induce a more immobilized component of the electron paramagnetic resonance spectrum, implying closing of the nucleotide site. The MT-bound states of all of the triphosphate analogs reveal two novel spectral components. The equilibrium between these two components is only weakly dependent on temperature. Both components have more restricted mobility than observed in MT-bound diphosphate states. Thus, the closing of the nucleotide pocket when the diphosphate state binds to MTs is amplified in the triphosphate state, perhaps promoting accelerated ATP hydrolysis. Consistent with this idea, molecular dynamics simulations show a good correlation between our spectroscopic data, X-ray crystallography, and the electron microscopy of MT-bound triphosphate-analog states.     

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.     

Modulation of the Kinesin ATPase Cycle by Neck Linker Docking and Microtubule Binding

Kinesin motor proteins use an ATP hydrolysis cycle to perform various functions in eukaryotic cells. Many questions remain about how the kinesin mechanochemical ATPase cycle is fine-tuned for specific work outputs. In this study, we use isothermal titration calorimetry and stopped-flow fluorometry to determine and analyze the thermodynamics of the human kinesin-5 (Eg5/KSP) ATPase cycle. In the absence of microtubules, the binding interactions of kinesin-5 with both ADP product and ATP substrate involve significant enthalpic gains coupled to smaller entropic penalties. However, when the wild-type enzyme is titrated with a non-hydrolyzable ATP analog or the enzyme is mutated such that it is able to bind but not hydrolyze ATP, substrate binding is 10-fold weaker than ADP binding because of a greater entropic penalty due to the structural rearrangements of switch 1, switch 2, and loop L5 on ATP binding. We propose that these rearrangements are reversed upon ATP hydrolysis and phosphate release. In addition, experiments on a truncated kinesin-5 construct reveal that upon nucleotide binding, both the N-terminal cover strand and the neck linker interact to modulate kinesin-5 nucleotide affinity. Moreover, interactions with microtubules significantly weaken the affinity of kinesin-5 for ADP without altering the affinity of the enzyme for ATP in the absence of ATP hydrolysis. Together, these results define the energy landscape of a kinesin ATPase cycle in the absence and presence of microtubules and shed light on the role of molecular motor mechanochemistry in cellular microtubule dynamics.