Monomeric Motor Research Articles

Microtubule-binding-induced allostery triggers LIS1 dissociation from dynein prior to cargo transport.

The lissencephaly-related protein LIS1 is a critical regulator of cytoplasmic dynein that governs motor function and intracellular localization (for example, to microtubule plus-ends). Although LIS1 binding is required for dynein activity, its unbinding prior to initiation of cargo transport is equally important, since preventing dissociation leads to dynein dysfunction. To understand whether and how dynein-LIS1 binding is modulated, we engineered dynein mutants locked in a microtubule-bound (MT-B) or microtubule-unbound (MT-U) state. Whereas the MT-B mutant exhibits low LIS1 affinity, the MT-U mutant binds LIS1 with high affinity, and as a consequence remains almost irreversibly associated with microtubule plus-ends. We find that a monomeric motor domain is sufficient to exhibit these opposing LIS1 affinities, and that this is evolutionarily conserved between yeast and humans. Three cryo-EM structures of human dynein with and without LIS1 reveal microtubule-binding induced conformational changes responsible for this regulation. Our work reveals key biochemical and structural insight into LIS1-mediated dynein activation.

Intracellular cargo transport by single-headed kinesin motors

Kinesin motor proteins that drive intracellular transport share an overall architecture of two motor domain-containing subunits that dimerize through a coiled-coil stalk. Dimerization allows kinesins to be processive motors, taking many steps along the microtubule track before detaching. However, whether dimerization is required for intracellular transport remains unknown. Here, we address this issue using a combination of in vitro and cellular assays to directly compare dimeric motors across the kinesin-1, -2, and -3 families to their minimal monomeric forms. Surprisingly, we find that monomeric motors are able to work in teams to drive peroxisome dispersion in cells. However, peroxisome transport requires minimal force output, and we find that most monomeric motors are unable to disperse the Golgi complex, a high-load cargo. Strikingly, monomeric versions of the kinesin-2 family motors KIF3A and KIF3B are able to drive Golgi dispersion in cells, and teams of monomeric KIF3B motors can generate over 8 pN of force in an optical trap. We find that intracellular transport and force output by monomeric motors, but not dimeric motors, are significantly decreased by the addition of longer and more flexible motor-to-cargo linkers. Together, these results suggest that dimerization of kinesin motors is not required for intracellular transport; however, it enables motor-to-motor coordination and high force generation regardless of motor-to-cargo distance. Dimerization of kinesin motors is thus critical for cellular events that require an ability to generate or withstand high forces.

Myosin Id localizes in dendritic spines through the tail homology 1 domain

Dendritic spines, the postsynaptic compartments at excitatory synapses, are capable of changing their shape and size to modulate synaptic transmission. The actin cytoskeleton and a variety of actin-binding proteins play a critical role in the dynamics of dendritic spines. Class I myosins are monomeric motor proteins that move along actin filaments using the energy of ATP hydrolysis. Of these class I myosins, myosin Id, the mammalian homolog of Drosophila Myo31DF, has been reported to be expressed in neurons, whereas its subcellular localization in neurons remained unknown. Here, we investigated the subcellular localization of myosin Id and determined the domain responsible for it. We found that myosin Id is enriched in the F-actin-rich pseudopodia of HEK293T cells and in the dendritic spines of primary hippocampal neurons. Both deletion and substitution of the tail homology 1 (TH1) domain drastically diminishes its colocalization with F-actin. In addition, the mutant form lacking the TH1 domain is less distributed in dendritic spines than is the full-length form. Taken together, our findings reveal that myosin Id localizes in dendritic spines through the TH1 domain.

The shaker-1 mouse myosin VIIa deafness mutation results in a severely reduced rate of the ATP hydrolysis step

Mutations in the MYO7A gene, encoding the motor protein myosin VIIa, can cause Usher 1B, a deafness/blindness syndrome in humans, and the shaker-1 phenotype, characterized by deafness, head tossing, and circling behavior, in mice. Myosin VIIa is responsible for tension bearing and the transduction mechanism in the stereocilia and for melanosome transport in the retina, in line with the phenotypic outcomes observed in mice. However, the effect of the shaker-1 mutation, a R502P amino acid substitution, on the motor function is unclear. To explore this question, we determined the kinetic properties and the effect on the filopodial tip localization of the recombinant mouse myosin VIIa-5IQ-SAH R502P (myoVIIa-sh1) construct. Interestingly, although residue 502 is localized to a region thought to be involved in interacting with actin, the kinetic parameters for actin binding changed only slightly for the mutant construct. However, the rate constant for ATP hydrolysis (k+H + k-H) was reduced by ∼200-fold from 12 s-1 to 0.05 s-1, making the hydrolysis step the rate-limiting step of the ATPase cycle in the presence and absence of actin. Given that wild-type mouse myosin VIIa is a slow, high-duty ratio, monomeric motor, this altered hydrolysis rate would reduce activity to extremely low levels. Indeed, the translocation to the filopodial tips was hampered by the diminished motor function of a dimeric construct of the shaker-1 mutant. We conclude that the diminished motor activity of this mutant is most likely responsible for impaired hearing in the shaker-1 mice.

Intracellular Cargo Transport by Single-Headed Kinesin Monomers

Kinesin motor proteins that drive intracellular transport share an overall architecture of two motor domain-containing subunits that dimerize through a coiled-coil stalk. Dimerization allows kinesins to be processive motors, taking many steps along the microtubule track before detaching. However, whether dimerization is required for intracellular transport remains unknown. Here, we address this issue using a combination of in vitro and cellular assays to directly compare dimeric motors across the kinesin-1, -2, and -3 families to their minimal monomeric forms. Surprisingly, we find that monomeric motors are able to work in teams to drive peroxisome dispersion in cells. However, peroxisome transport requires minimal force output, and we find that most monomeric motors are unable to disperse the Golgi complex, a high-load cargo. Strikingly, monomeric versions of the kinesin-2 family motors KIF3A and KIF3B are able to drive Golgi dispersion in cells, and teams of monomeric KIF3B motors can generate over 8 pN of force in an optical trap. We find that intracellular transport and force output by monomeric motors, but not dimeric motors, are significantly decreased by the addition of longer and more flexible motor-to-cargo linkers. Together, these results suggest that dimerization of kinesin motors is not required for intracellular transport; however, it enables motor-to-motor coordination and high force generation regardless of motor-to-cargo distance. Dimerization of kinesin motors is thus critical for cellular events that require an ability to generate or withstand high forces.

Loss of function of KIF1B impairs oocyte meiotic maturation and early embryonic development in mice

Kinesin family member 1B (KIF1B) is an important microtubule-dependent monomeric motor in mammals, although little is known about its role in meiosis. We profiled KIF1B expression and localization during oocyte maturation and early embryonic development in mice, revealing a dynamic pattern throughout meiotic progression. Depletion or inhibition of KIF1B leads to abnormal polar body extrusion, disordered spindle dynamics, defects in chromosome congression, increased aneuploidy, and impaired embryonic development. Further, KIF1B depletion affects the distribution of mitochondria and abundance of ATP. Taken together, our study demonstrates that mouse KIF1B is important for spindle assembly, chromosome congression, and mitochondrial distribution during oocyte maturation and early embryonic development. Mol. Reprod. Dev. 83: 1027-1040, 2016 © 2016 Wiley Periodicals, Inc.

Motor and Tail Homology 1 (TH1) Domains Antagonistically Control Myosin-1 Dynamics

Class 1 myosins are monomeric motor proteins that fulfill diverse functions at the membrane/cytoskeletal interface. All myosins-1 contain a motor domain, which binds actin, hydrolyzes ATP, and generates forces, and a TH1 domain, which interacts directly with membrane lipids. In most cases, TH1 is needed for proper subcellular localization and presumably function, although little is known about how this domain regulates the behavior of class 1 myosins in live cells. To address this, we used single molecule total internal reflection fluorescence microscopy to examine the dynamics of the well-characterized myosin-1a isoform during interactions with the cortex of living cells. Our studies revealed that full-length myosin-1a exhibits restricted mobility relative to TH1 alone. Motor domain mutations that disrupt actin binding increased the mobility of full-length myosin-1a, whereas mutations to the TH1 domain that are known to reduce steady-state targeting to the plasma membrane unexpectedly reduced mobility. Deletion of the calmodulin-binding lever arm in Myo1a mimicked the impact of actin-binding mutations. Finally, myosin-1b, which demonstrates exquisite sensitivity to mechanical load, exhibited dynamic behavior nearly identical to myosin-1a. These studies are the first, to our knowledge, to explore class 1 myosin dynamics at the single-molecule level in living cells; our results suggest a model where the motor domain restricts dynamics via a mechanism that requires the lever arm, whereas the TH1 domain allows persistent diffusion in close proximity to the plasma membrane.

Leptin-mediated regulation of MT1-MMP localization is KIF1B dependent and enhances gastric cancer cell invasion

Leptin overexpression is closely correlated with gastric cancer (GC) invasion, but its exact effect and the underlying mechanism in tumorigenesis remain poorly understood. Membrane type 1-matrix metalloproteinase (MT1-MMP), a surface-anchored 'master switch' proteinase, is overexpressed and plays crucial roles in tumor invasion. Here, we characterized the influence of leptin on the generation and surface localization of MT1-MMP in GC and elucidated its molecular mechanisms. Our results revealed that leptin promoted GC cell invasion in vitro by upregulating MT1-MMP expression. Furthermore, cell surface biotinylation assay and flow cytometry demonstrated that the surface expression of MT1-MMP was also enhanced by leptin, and knockdown of kinesin family member 1B (KIF1B, a microtubule plus end-directed monomeric motor protein) by small interference RNA inhibited this process. Notably, coimmunoprecipitation analysis indicated that leptin enhanced the interaction of MT1-MMP with KIF1B in a time-dependent manner, which consequently contributed to GC cell invasion. Moreover, leptin increased MT1-MMP or KIF1B expression by the protein kinase B (AKT) pathway and extracellular signal-regulated kinase 1/2 partially participated in this process. However, only AKT was implicated in the leptin-mediated membrane localization of MT1-MMP. Immunohistochemistry analysis revealed that leptin, MT1-MMP and KIF1B are overexpressed in GC tissues, and they positively correlated with clinical stage and lymph node metastasis. These observations indicate that this regulatory network exists in vivo. Taken together, our findings suggest that leptin is an effective intracellular stimulator of MT1-MMP and that leptin-enhanced cell surface localization of MT1-MMP is dependent on KIF1B, which consequently plays a critical role in GC invasion.

Myosin-1A Targets to Microvilli Using Multiple Membrane Binding Motifs in the Tail Homology 1 (TH1) Domain

One of the most abundant components of the enterocyte brush border is the actin-based monomeric motor, myosin-1a (Myo1a). Within brush border microvilli, Myo1a carries out a number of critical functions at the interface between membrane and actin cytoskeleton. Proper physiological function of Myo1a depends on its ability to bind to microvillar membrane, an interaction mediated by a C-terminal tail homology 1 (TH1) domain. However, little is known about the mechanistic details of the Myo1a-TH1/membrane interaction. Structure-function analysis of Myo1a-TH1 targeting in epithelial cells revealed that an N-terminal motif conserved among class I myosins and a C-terminal motif unique to Myo1a-TH1 are both required for steady state microvillar enrichment. Purified Myo1a bound to liposomes composed of phosphatidylserine and phosphoinositol 4,5-bisphosphate, with moderate affinity in a charge-dependent manner. Additionally, peptides of the N- and C-terminal regions required for targeting were able to compete with Myo1a for binding to highly charged liposomes in vitro. Single molecule total internal reflection fluorescence microscopy showed that these motifs are also necessary for slowing the membrane detachment rate in cells. Finally, Myo1a-TH1 co-localized with both lactadherin-C2 (a phosphatidylserine-binding protein) and PLCδ1-PH (a phosphoinositol 4,5-bisphosphate-binding protein) in microvilli, but only lactaderin-C2 expression reduced brush border targeting of Myo1a-TH1. Together, our results suggest that Myo1a targeting to microvilli is driven by membrane binding potential that is distributed throughout TH1 rather than localized to a single motif. These data highlight the diversity of mechanisms that enable different class I myosins to target membranes in distinct biological contexts.

Mycobacterium smegmatis RqlH defines a novel clade of bacterial RecQ-like DNA helicases with ATP-dependent 3′–5′ translocase and duplex unwinding activities

The Escherichia coli RecQ DNA helicase participates in a pathway of DNA repair that operates in parallel to the recombination pathway driven by the multisubunit helicase–nuclease machine RecBCD. The model mycobacterium Mycobacterium smegmatis executes homologous recombination in the absence of its helicase–nuclease machine AdnAB, though it lacks a homolog of E. coli RecQ. Here, we identify and characterize M. smegmatis RqlH, a RecQ-like helicase with a distinctive domain structure. The 691-amino acid RqlH polypeptide consists of a RecQ-like ATPase domain (amino acids 1–346) and tetracysteine zinc-binding domain (amino acids 435–499), separated by an RqlH-specific linker. RqlH lacks the C-terminal HRDC domain found in E. coli RecQ. Rather, the RqlH C-domain resembles bacterial ComF proteins and includes a phosphoribosyltransferase-like module. We show that RqlH is a DNA-dependent ATPase/dATPase that translocates 3′–5′ on single-stranded DNA and has 3′–5′ helicase activity. These functions inhere to RqlH-(1–505), a monomeric motor unit comprising the ATPase, linker and zinc-binding domains. RqlH homologs are distributed widely among bacterial taxa. The mycobacteria that encode RqlH lack a classical RecQ, though many other Actinobacteria have both RqlH and RecQ. Whereas E. coli K12 encodes RecQ but lacks a homolog of RqlH, other strains of E. coli have both RqlH and RecQ.

Two single-headed myosin V motors bound to a tetrameric adapter protein form a processive complex

Myo4p, one of two class V myosins in budding yeast, continuously transports messenger RNA (mRNA) cargo in the cell but is nonprocessive when characterized in vitro. The adapter protein She3p tightly binds to the Myo4p rod, forming a single-headed motor complex. In this paper, we show that two Myo4p-She3p motors are recruited by the tetrameric mRNA-binding protein She2p to form a processive double-headed complex. The binding site for She3p was mapped to a single α helix that protrudes at right angles from She2p. Processive runs of several micrometers on yeast actin-tropomyosin filaments were observed only in the presence of She2p, and, thus, motor activity is regulated by cargo binding. While moving processively, each head steps ~72 nm in a hand-over-hand motion. Coupling two high-duty cycle monomeric motors via a common cargo-binding adapter protein creates a complex with transport properties comparable with a single dimeric processive motor such as vertebrate myosin Va.

Conformational changes, diffusion and collective behavior in monomeric kinesin-based motility

Molecular motors convert chemical energy into mechanical motion and power the transport of material within living cells; the motion of a motor is thought to be influenced by stochastic chemical state transitions of the molecule as well as intramolecular diffusion of one motor head seeking the next binding site. Existing models for the motility of single-headed monomeric motors that map the system to a simplified two-state Brownian ratchet have some predictive power, but in general are unable to elucidate the contributions of different molecular level processes to the overall effective parameters. In this work, we build a detailed molecular level model of monomeric kinesin motility that naturally incorporates conformational changes (power strokes) and biased diffusion. Our results predict that mean velocity is most sensitive to the power stroke size, while run length distribution is sensitive primarily to the strength of the microtubule bias potential with a weak dependence on power stroke that can be tuned by the strength of an applied load. In addition, we demonstrate that motor pairs attached to the same cargo can cooperatively function to increase motility in both the plus- and minus-end directions. These findings illustrate the importance of a detailed mechanochemical model for dissecting the contributions of microscopic parameters to monomeric kinesin dynamics.

Multiple Myo4 motors enhance ASH1 mRNA transport in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, ASH1 mRNA is transported to the bud tip by the class V myosin Myo4. In vivo, Myo4 moves RNA in a rapid and continuous fashion, but in vitro Myo4 is a nonprocessive, monomeric motor that forms a complex with She3. To understand how nonprocessive motors generate continuous transport, we used a novel purification method to show that Myo4, She3, and the RNA-binding protein She2 are the sole major components of an active ribonucleoprotein transport unit. We demonstrate that a single localization element contains multiple copies of Myo4 and a tetramer of She2, which suggests that She2 may recruit multiple motors to an RNA. Furthermore, we show that increasing the number of Myo4-She3 molecules bound to ASH1 RNA in the absence of She2 increases the efficiency of RNA transport to the bud. Our data suggest that multiple, nonprocessive Myo4 motors can generate continuous transport of mRNA to the bud tip.

Myosin-1 Function Extends to Microtubule-Dependent Processes

Class 1 myosins are motor proteins found on various organelles and at defined structures at the cell periphery, where they play important roles in organelle translocation, intracellular transport, and cytoskeleton organization. Here, we investigated the cellular function of Dictyostelium myosin-1C, a long-tailed, monomeric motor with three tail homology domains called TH1, TH2, and TH3. We identified that in addition to its actin-dependent function in endocytotic processes during interphase, in Dictyostelium cells the motor co-localizes with centrosomes and spindle microtubules (MT) during mitosis. In vitro TIRF microscopy experiments and MT-cosedimentation assays using truncated myosin-1C tail constructs revealed a direct binding of myosin-1C tail to MTs. Only constructs containing TH1 and TH2 bound efficiently to MTs. Moreover, these two domains were sufficient to prevent MT depolymerization at low nanomolar concentrations of myosin-1C tail, while MT formation, i.e. nucleation and elongation, was unaffected. Additionally, we observed myosin-1C tail mediated cross-linking of MTs to F-actin. In cells, myosin-1C constructs lacking the motor domain did not associate with the spindle. This demonstrates that actin-dependent motor function is required for the cell cycle-dependent re-localization of myosin-1C from actin-rich structures at the cell periphery to MT-associated mitotic structures. Cells producing a hydrolysis deficient full-length myosin-1C mutant exhibited reduced growth rates with increased size of nuclei due to defects in spindle alignment and prolonged mitosis. Single kinesin molecule motility assays showed that MT-bound myosin-1C reduced the attachment rate of kinesin-1 to MTs without affecting its velocity and run-length. From this we propose that myosin-1C may regulate MT-dependent motility and MT dynamics. Our cell biological and functional characterization of a long-tailed class 1 myosin shows that myosin function is not limited to the F-actin network but extends to MT associated processes.

Dimeric Centromere Protein E (CENP-E) Promotes Microtubule-Elongation at the Plus-Ends Of Microtubules

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis including establishment of microtubule (MT)-chromosome linkage and processive movement of monooriented chromosomes on MTs for proper alignment at metaphase. Fluorescence microscopy studies were performed to test the hypothesis that CENP-E promotes MT-elongation at the MT plus-ends. CENP-E constructs were engineered, expressed, and purified which yielded dimeric and monomeric motor proteins. The results show that dimeric CENP-E promotes plus-end directed MT gliding at 11 ± 0.005 nm/sec (n=173 MTs). Real-time microscopy assays were performed to image CENP-E promoted elongation of GMPcPP-stabilized polarity marked FITC MTs. The results revealed that out of the 270 polarity marked MTs examined, 164 MTs (60%) exhibited CENP-E promoted MT plus-end extension by GTP-tubulin (1.48 ± 0.37 μm/30 min; n=200 MTs) in the presence of MgATP. In contrast, dimeric Kinesin-1, dimeric Eg5, and CENP-E in the presence of AMPPNP did not show this pronounced MT elongation. These results suggest that CENP-E as part of its function for chromosome kinetochore attachment to MTs plays a direct role in kinetochore MT extension during congression. Supported by NIH GM54141 to Susan P. Gilbert.

The mechanisms of kinesin motor motility: lessons from the monomeric motor KIF1A

Most kinesins move processively along microtubules by using energy derived from ATP hydrolysis. Almost all of the intermediate structures of this ATPase reaction cycle have been solved for the monomeric kinesin 3 family motor KIF1A. Based on this structural information, we propose a common mechanism of kinesin motility, focusing on the regulation of kinesin motility through their interaction with microtubules and by their 'neck-linker' region, which connects their motor domain to cargo and kinesin partner heads.

Ste20-kinase-dependent TEDS-site phosphorylation modulates the dynamic localisation and endocytic function of the fission yeast class I myosin, Myo1

Type I myosins are monomeric motors involved in a range of motile and sensory activities in different cell types. In simple unicellular eukaryotes, motor activity of class I myosins is regulated by phosphorylation of a conserved 'TEDS site' residue within the motor domain. The mechanism by which this phosphorylation event affects the cellular function of each myosin I remains unclear. The fission yeast myosin I, Myo1, activates Arp2/3-dependent polymerisation of cortical actin patches and also regulates endocytosis. Using mutants and Myo1-specific antibodies, we show that the phosphorylation of the Myo1 TEDS site (serine 361) plays a crucial role in regulating this protein's dynamic localisation and cellular function. We conclude that although phosphorylation of serine 361 does not affect the ability of this motor protein to promote actin polymerisation, it is required for Myo1 to recruit to sites of endocytosis and function during this process.

Lattice Structure of Cytoplasmic Microtubules in a Cultured Mammalian Cell

Tubulin can polymerize in two distinct arrangements: “B-lattices,” in which the α-tubulins of one protofilament lie next to α-tubulins in the neighboring protofilaments, or the “A” configuration, where α-tubulins lie beside β-tubulins. Microtubules (MTs) in flagellar axonemes and those assembled from pure tubulin in vitro display only B-lattices, but recent work shows that A-lattices are found when tubulin co-polymerizes in vitro with an allele of end-binding protein 1 that lacks C-terminal sequences. This observation suggests that cytoplasmic MTs, which form in the presence of this “tip-associating protein,” may have A-lattices. To test this hypothesis, we have decorated interphase MTs in 3T3 cells with monomeric motor domains from the kinesin-like protein Eg5. These MTs show only B-lattices, as confirmed by visual inspection of electron cryo-tomograms and power spectra of single projection views, imaged at higher electron dose. This result is significant because 13 protofilament MTs with B-lattices must include a “seam,” one lateral domain where adjacent dimers are in the A-configuration. It follows that cytoplasmic MTs are not cylindrically symmetric; they have two distinct faces, which may influence the binding patterns of functionally significant MT-interacting proteins.

Dimeric Centromere Protein E (CENP-E) Promotes Microtubule-elongation At The Plus-ends Of Microtubules

Centromere protein CENP-E is a dimeric kinesin (Kinesin-7 family) with critical roles in mitosis including establishment of microtubule (MT)-chromosome linkage, processive movement of monooriented chromosomes on MTs for proper alignment at metaphase, and as a tension sensor at mitotic checkpoint to signal onset to anaphase. Fluorescence microscopy studies were performed to test the hypothesis that CENP-E promotes MT-elongation at the MT plus-ends. CENP-E constructs were engineered, expressed, and purified which yielded dimeric and monomeric motor proteins. The results show that dimeric CENP-E promotes plus-end directed MT gliding at 11 ± 0.005 nm/sec (n=173 MTs). Real-time microscopy assays were performed to image CENP-E promoted elongation of GMPcPP-stabilized polarity marked FITC MTs. The results revealed that out of the 270 polarity marked MTs examined, 164 MTs (60%) exhibited CENP-E promoted MT plus-end extension by GTP-tubulin (1.48 ± 0.37 μm/30 min; n=200 MTs) in the presence of MgATP. In contrast, dimeric Kinesin-1, dimeric Eg5, and CENP-E in the presence of AMPPNP did not show this pronounced MT elongation. These results suggest that CENP-E as part of its function for chromosome kinetochore attachment to MTs plays a direct role in kinetochore MT extension during congression. Supported by NIH GM54141 and NIH Career Development Award K02-AR47841 to SPG.

Millisecond Time-lapsed Monitoring of ATP Hydrolysis by Human Eg5 Kinesin: Real-time Dynamics of Conformation and Chemistry in vitro

The hydrolysis of ATP is one of the key chemical reactions in life. Its mechanistic details in biological systems have been a challenge to unravel, as chemical changes in proteins during ATP hydrolysis go hand-in-hand with a series of large-scale conformational alterations. Monitoring the dynamics of chemistry and structure has been experimentally intractable, and requires application of novel methods. Herein, we present time-lapsed monitoring of an in vitro ATP hydrolysis reaction by a kinesin motor protein with 180-millisecond time resolution. Kinesin proteins are one of three major categories of motor proteins, capable of using ATP hydrolysis to power force generation and subsequent movement along cytoskeletal elements in cells. Our model protein system is human Eg5 (HsEg5), a Kinesin-5 motor protein participating in the spindle pole segregation during mitosis in higher eukaryotes. Truncated to its monomeric motor domain, we purified active HsEg5 and confirmed its ability to hydrolyze ATP. To monitor dynamic structural and chemical changes during ATP hydrolysis concomitantly, we used difference Fourier-transform infrared (FTIR) spectroscopy with HsEg5 kinesin samples, triggering initiation of the ATPase reaction by UV-photolysis of caged ATP. Interpretation of these biological data was guided by model compound data on ATP derivatives. The time-lapse data highlighted resolution of two distinct sets of conformational changes: a series of HsEg5 structural changes that precedes ATP hydrolysis and a set of structural alterations that occurs upon onset of ATP hydrolysis. Thus, we conclude that we have the first direct observation of dynamic conformational changes caused by the ATP binding in any kinesin motor protein. Secondly, the structural modifications that occur HsEg5 when ATP hydrolysis is initiated are different than those in the substrate-binding step.