A kinesin mutation that uncouples motor domains and desensitizes the gamma-phosphate sensor

Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr(291) of Drosophila kinesin heavy chain is identical in all superfamily members and is located in alpha-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr(291) results in complete loss of function in vivo. In vitro, the T291M mutation disrupts the ATPase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514). The pre-steady-state kinetic analysis presented here shows that ATP binding is weakened significantly, and the rate of ATP hydrolysis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the gamma-phosphate have been altered. The results indicate that there is a signaling defect between the motor domains of the T291M dimer. The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more independently rather than in the strict, coordinated fashion that is typical of kinesin.     

Motor domain mutation traps kinesin as a microtubule rigor complex

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

Nucleotide specificity of the enzymatic and motile activities of dynein, kinesin, and heavy meromyosin

The substrate specificities of dynein, kinesin, and myosin substrate turnover activity and cytoskeletal filament-driven translocation were examined using 15 ATP analogues. The dyneins were more selective in their substrate utilization than bovine brain kinesin or muscle heavy meromyosin, and even different types of dyneins, such as 14S and 22S dynein from Tetrahymena cilia and the beta-heavy chain-containing particle from the outer-arm dynein of sea urchin flagella, could be distinguished by their substrate specificities. Although bovine brain kinesin and muscle heavy meromyosin both exhibited broad substrate specificities, kinesin-induced microtubule translocation varied over a 50-fold range in speed among the various substrates, whereas heavy meromyosin-induced actin translocation varied only by fourfold. With both kinesin and heavy meromyosin, the relative velocities of filament translocation did not correlate well with the relative filament-activated substrate turnover rates. Furthermore, some ATP analogues that did not support the filament translocation exhibited filament-activated substrate turnover rates. Filament-activated substrate turnover and power production, therefore, appear to become uncoupled with certain substrates. In conclusion, the substrate specificities and coupling to motility are distinct for different types of molecular motor proteins. Such nucleotide "fingerprints" of enzymatic activities of motor proteins may prove useful as a tool for identifying what type of motor is involved in powering a motility-related event that can be reconstituted in vitro.     

Microtubule-kinesin interface mutants reveal a site critical for communication

Strict coordination of the two motor domains of kinesin is required for driving the processive movement of organelles along microtubules. Glutamate 164 of the kinesin heavy chain was shown to be critical for kinesin function through in vivo genetics in Drosophila melanogaster. The mutant motor E164K exhibited reduced steady-state ATPase activity and higher affinity for both ATP and microtubules. Moreover, an alanine substitution at this position (E164A) caused similar defects. It became stalled on the microtubule and was unable to bind and hydrolyze ATP at the second motor domain. Glu(164), which has been conserved through evolution, is located at the motor-microtubule interface close to key residues on helix alpha12 of beta-tubulin. We explored further the contributions of Glu(164) to motor function using several site-directed mutant proteins: E164K, E164N, E164D, E164Q, and D165A. The results indicate that the microtubule-E164K complex can only bind and hydrolyze one ATP. ATP with increased salt was able to dissociate a population of E164K motors from the microtubule but could not dissociate E164A. We tested the basis of the stabilized microtubule interaction with E164K, E164N, and E164A. The results provide new insights about the motor-microtubule interface and the pathway of communication for processive motility.     

A kinesin heavy chain (KIF5A) mutation in hereditary spastic paraplegia (SPG10)

We have identified a missense mutation in the motor domain of the neuronal kinesin heavy chain gene KIF5A, in a family with hereditary spastic paraplegia. The mutation occurs in the family in which the SPG10 locus was originally identified, at an invariant asparagine residue that, when mutated in orthologous kinesin heavy chain motor proteins, prevents stimulation of the motor ATPase by microtubule-binding. Mutation of kinesin orthologues in various species leads to phenotypes resembling hereditary spastic paraplegia. The conventional kinesin motor powers intracellular movement of membranous organelles and other macromolecular cargo from the neuronal cell body to the distal tip of the axon. This finding suggests that the underlying pathology of SPG10 and possibly of other forms of hereditary spastic paraplegia may involve perturbation of neuronal anterograde (or retrograde) axoplasmic flow, leading to axonal degeneration, especially in the longest axons of the central nervous system.     

Crystal structure of kinesin regulated by Ca(2+)-calmodulin

Kinesins orchestrate cell division by controlling placement of chromosomes. Kinesins must be precisely regulated or else cell division fails. Calcium, a universal second messenger in eukaryotes, and calmodulin regulate some kinesins by causing the motor to dissociate from its biological track, the microtubule. Our focus was the mechanism of calcium regulation of kinesin at atomic resolution. Here we report the crystal structure of kinesin-like calmodulin-binding protein (KCBP) from potato, which was resolved to 2.3 A. The structure reveals three subdomains of the regulatory machinery located at the C terminus extension of the kinesin motor. Calmodulin that is activated by Ca2+ ions binds to an alpha-helix positioned on the microtubule-binding face of kinesin. A negatively charged segment following this helix competes with microtubules. A mimic of the conventional kinesin neck, connecting the calmodulin-binding helix to the KCBP motor core, links the regulatory machine to the kinesin catalytic cycle. Together with biochemical data, the crystal structure suggests that Ca(2+)-calmodulin inhibits the binding of KCBP to microtubules by blocking the microtubule-binding sites on KCBP.     

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin’s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.     

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.     

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.     

Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport

In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end- directed movement. The latter was selectively blocked in the rigor- mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo.     

Nucleotide-dependent movements of the kinesin motor domain predicted by simulated annealing.

The structure of an ATP-bound kinesin motor domain is predicted and conformational differences relative to the known ADP-bound form of the protein are identified. The differences should be attributed to force-producing ATP hydrolysis. Candidate ATP-kinesin structures were obtained by simulated annealing, by placement of the ATP gamma-phosphate in the crystal structure of ADP-kinesin, and by interatomic distance constraints. The choice of such constraints was based on mutagenesis experiments, which identified Gly-234 as one of the gamma-phosphate sensing residues, as well as on structural comparison of kinesin with the homologous nonclaret disjunctional (ncd) motor and with G-proteins. The prediction of nucleotide-dependent conformational differences reveals an allosteric coupling between the nucleotide pocket and the microtubule binding site of kinesin. Interactions of ATP with Gly-234 and Ser-202 trigger structural changes in the motor domain, the nucleotide acting as an allosteric modifier of kinesin's microtubule-binding state. We suggest that in the presence of ATP kinesin's putative microtubule binding regions L8, L12, L11, alpha4, alpha5, and alpha6 form a face complementary in shape to the microtubule surface; in the presence of ADP, the microtubule binding face adopts a more convex shape relative to the ATP-bound form, reducing kinesin's affinity to the microtubule.     

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

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

Structural dynamics of the microtubule binding and regulatory elements in the kinesin-like calmodulin binding protein

Kinesins are molecular motors that power cell division and transport of various proteins and organelles. Their motor activity is driven by ATP hydrolysis and depends on interactions with microtubule tracks. Essential steps in kinesin movement rely on controlled alternate binding to and detaching from the microtubules. The conformational changes in the kinesin motors induced by nucleotide and microtubule binding are coordinated by structural elements within their motor domains. Loop L11 of the kinesin motor domain interacts with the microtubule and is implicated in both microtubule binding and sensing nucleotide bound to the active site of kinesin. Consistent with its proposed role as a microtubule sensor, loop L11 is rarely seen in crystal structures of unattached kinesins. Here, we report four structures of a regulated plant kinesin, the kinesin-like calmodulin binding protein (KCBP), determined by X-ray crystallography. Although all structures reveal the kinesin motor in the ATP-like conformation, its loop L11 is observed in different conformational states, both ordered and disordered. When structured, loop L11 adds three additional helical turns to the N-terminal part of the following helix α4. Although interactions with protein neighbors in the crystal support the ordering of loop L11, its observed conformation suggests the conformation for loop L11 in the microtubule-bound kinesin. Variations in the positions of other features of these kinesins were observed. A critical regulatory element of this kinesin, the calmodulin binding helix positioned at the C-terminus of the motor domain, is thought to confer negative regulation of KCBP. Calmodulin binds to this helix and inserts itself between the motor and the microtubule. Comparison of five independent structures of KCBP shows that the positioning of the calmodulin binding helix is not decided by crystal packing forces but is determined by the conformational state of the motor. The observed variations in the position of the calmodulin binding helix fit the regulatory mechanism previously proposed for this kinesin motor.     

Inhibitors of kinesin activity from structure-based computer screening

Kinesin motor proteins use ATP hydrolysis for transport along microtubules in the cell. We sought to identify small organic ligands to inhibit kinesin's activity. Candidate molecules were identified by computational docking of commercially available compounds using the computer program DOCK. Compounds were docked at either of two sites, and a selection was tested for inhibition of microtubule-stimulated ATPase activity. Twenty-two submillimolar inhibitors were identified. Several inhibitors appeared to be competitive for microtubule binding and not for ATP binding, and three compounds showed 50% inhibition down to single-digit micromolar levels. Most inhibitors grouped into four distinct classes (fluoresceins, phenolphthaleins, anthraquinones, and naphthylene sulfonates). We measured the binding of one inhibitor, rose bengal lactone (RBL), to kinesin (dissociation constant 2.5 microM) by its increase in steady-state fluorescence anisotropy. The RBL binding site on kinesin was localized by fluorescent resonance energy transfer (FRET) using a donor fluorophore (coumarin) covalently attached at unique, surface-exposed cysteine residues engineered at positions 28, 149, 103, 220, or 330. RBL was found to bind in its original docked site: the pocket cradled by loop 8 and beta-strand 5 in kinesin's three-dimensional structure. These results confirm this region's role in microtubule binding and identify this pocket as a novel binding site for kinesin inhibition.     

Kinesin's IAK tail domain inhibits initial microtubule-stimulated ADP release

Kinesin undergoes a global folding conformational change from an extended active conformation at high ionic concentrations to a compact inhibited conformation at physiological ionic concentrations. Here we show that much of the observed ATPase activity of folded kinesin is due to contamination with proteolysis fragments that can still fold, but retain an activated ATPase function. In contrast, kinesin that contains an intact IAK-homology region exhibits pronounced inhibition of its ATPase activity (140-fold in 50 mM KCl) and weak net affinity for microtubules in the presence of ATP, resulting from selective inhibition of the release of ADP upon initial interaction with a microtubule. Subsequent processive cycling is only partially inhibited. Fusion proteins containing residues 883-937 of the kinesin alpha-chain bind tightly to microtubules; exposure of this microtubule-binding site in proteolysed species is probably responsible for their activated ATPase activities at low microtubule concentrations.     

Isolation of a 45-kDa fragment from the kinesin heavy chain with enhanced ATPase and microtubule-binding activities

Kinesin is a microtubule-activated, mechanochemical ATPase capable of moving particles along microtubules and making microtubules glide along a solid substrate. In this study we used limited proteolysis to study the structure of bovine brain kinesin, a heterotetramer composed of two heavy (120-kDa) and two light (62-kDa) chains. alpha-chymotrypsin, trypsin, and subtilisin all produced a protease-resistant 45-kDa fragment from the kinesin heavy chain. As isolated by gel-filtration chromatography, this fragment contains both the microtubule-binding site and the ATP catalytic site of the molecule. Proteolytic cleavage stimulated microtubule-dependent Mg2+-ATPase activity 4- to 5-fold up to 75-120 mumol ATP/min/mg. Cleavage also increased the affinity of the fragment for microtubules at least 10-fold. Since the purified fragment does not support the gliding of flagellar axonemes, we propose that cleavage of the heavy chain uncouples ATPase activity from its translocator activity, which may require other parts of the molecule.     

Working strokes by single molecules of the kinesin-related microtubule motor ncd

The ncd protein is a dimeric, ATP-powered motor that belongs to the kinesin family of microtubule motor proteins. Here we resolve single mechanochemical cycles of recombinant, dimeric, full-length ncd, using optical-tweezers-based instrumentation and a three-bead, suspended-microtubule assay. Under conditions of limiting ATP, isolated and transient microtubule-binding events exhibit exponentially distributed and ATP-concentration-dependent lifetimes. These events do not involve consecutive steps along the microtubule, quantitatively confirming that ncd is non-processive. At low loads, a single motor molecule produces ATP-triggered working strokes of about 9 nm, which occur at the ends of binding events.     

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.     

Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide

Kinesin was extensively purified from bovine brain cytosol by a microtubule-binding step in the presence of 5'-adenylyl imidodiphosphate (AMP-PNP), followed by gel filtration chromatography and sucrose gradient ultracentrifugation. The products consistently contained 124,000 (124K) and 64,000 (64K) dalton polypeptides. These two polypeptides appear to represent heavy and light chains of kinesin, respectively, because they copurified on sucrose gradients to a constant and equimolar stoichiometry and bound stably to microtubules in the presence of AMP-PNP but not ATP. The mobilities of 124K and 64K in sodium dodecyl sulfate-polyacrylamide gels under reducing conditions were the same as under nonreducing conditions. A diffusion coefficient of (2.24 +/- 0.21) X 10(-7) cm2 s-1 and a sedimentation coefficient of (9.56 +/- 0.34) X 10(-13) s were determined for native kinesin by gel filtration and sucrose gradient ultracentrifugation, respectively. These values were used to calculate a native molecular weight of about 379,000 and suggest that kinesin has an axial ratio of approximately 20. Extensively purified kinesin exhibited microtubule-activated ATPase activity, and only the 124K subunit incorporated ATP in photoaffinity labeling experiments using [32P]ATP. Collectively, these data favor the interpretation that bovine brain kinesin is a highly elongated, microtubule-activated ATPase comprising two subunits each of 124,000 and 64,000 daltons, that the subunits are not linked to one another by disulfide bonds, and that the heavy chains are the ATP-binding subunits.     

ATPase kinetic characterization and single molecule behavior of mutant human kinesin motors defective in microtubule-based motility

Conventional kinesin is a microtubule-based motor protein that is an important model system for understanding mechanochemical transduction. To identify regions of the kinesin protein that participate in microtubule binding and force production, Woehlke et al. [(1997) Cell 90, 207-216] generated 35 alanine mutations in solvent-exposed residues. Here, we have performed presteady-state kinetic and single molecule motility analyses on three of these mutants [Y138A, loop 11 triple (L248A/D249A/E250A), and E311A] that exhibited a similar approximately 3-fold reduction in both microtubule gliding velocity and microtubule-stimulated ATPase activity. All mutants showed normal second-order ATP binding kinetics, indicating correct folding of the active site. The Y138A and loop 11 triple mutants were defective both in nucleotide hydrolysis and in microtubule-stimulated ADP release rates, the latter suggesting a defect in allosteric communication between the microtubule and the active site. A single molecule fluorescence assay further revealed that the loop 11 mutant is defective in initiating processive motion, suggesting that this loop is important for the initial contact between kinesin and the microtubule. Y138A, on the other hand, can bind to the microtubule normally but cannot move processively. For E311A, neither the rate of nucleotide hydrolysis nor ADP release could account for its slower ATPase and gliding velocity, which suggests that either phosphate release or a conformational transition is rate-limiting in this mutant. The single molecule assay showed that E311A has a reduced velocity of movement, but is not defective in processivity. Thus, while these mutants behave similarly in solution ATPase and multiple motor gliding assays, kinetic and single molecule analyses reveal defects in distinct processes in kinesin's mechanochemical cycle.