ABSTRACT
Kinesins are P-loop NTPases that can do mechanical work. Like small G-proteins, to which they are related, kinesins execute a program of active site conformational changes that cleaves the terminal phosphate from an NTP substrate. But unlike small G-proteins, kinesins can amplify and harness these conformational changes in order to exert force. In this short review I summarize current ideas about how the kinesin active site works and outline how the active site chemistry is coupled to the larger-scale structural cycle of the kinesin motor domain. Focusing largely on kinesin-1, the best-studied kinesin, I discuss how the active site switch machinery of kinesin cycles between three distinct states, how docking of the neck linker stabilizes two of these states, and how tension-sensitive and position-sensitive neck linker docking may modulate both the hydrolysis step of ATP turnover and the trapping of product ADP in the active site. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 476-482, 2016.
Subject(s)
Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Kinesins/chemistry , Kinesins/metabolism , Animals , Catalytic Domain , Humans , Hydrolysis , Protein Structure, SecondaryABSTRACT
Eukaryotic systems self-organise by using molecular railways to shuttle specific sets of molecular components to specific locations. In this way, cells are enabled to become larger, more complex and more varied, subtle and effective in their activities. Because of the fundamental importance of molecular railways in eukaryotic systems, understanding how these railways work is an important research goal. Mechanochemical cell biology is a newly circumscribed subject area that concerns itself with the molecular and cell biological mechanisms of motorised directional transport in living systems.
Subject(s)
Cell Biology , Cytological Techniques/methods , Animals , Humans , Models, Biological , Stress, MechanicalABSTRACT
Kinesin is a molecular walking machine that organizes cells by hauling packets of components directionally along microtubules. The physical mechanism that impels directional stepping is uncertain. We show here that, under very high backward loads, the intrinsic directional bias in kinesin stepping can be reversed such that the motor walks sustainedly backwards in a previously undescribed mode of ATP-dependent backward processivity. We find that both forward and backward 8-nm steps occur on the microsecond timescale and that both occur without mechanical substeps on this timescale. The data suggest an underlying mechanism in which, once ATP has bound to the microtubule-attached head, the other head undergoes a diffusional search for its next site, the outcome of which can be biased by an applied load.
Subject(s)
Kinesins/metabolism , Molecular Motor Proteins/metabolism , Movement , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Algorithms , Animals , Biomechanical Phenomena , Diffusion , Drosophila , Microtubules/metabolism , Models, Biological , Time FactorsSubject(s)
Adenosine Diphosphate/metabolism , Kinesins/antagonists & inhibitors , Microtubules/metabolism , Microtubules/physiology , Pyrimidines/metabolism , Thiones/metabolism , Xenopus Proteins/antagonists & inhibitors , Animals , Kinesins/metabolism , Rats , Xenopus , Xenopus Proteins/metabolismABSTRACT
A new optical trapping study shows that the stepsize of cytoplasmic dynein varies according to the applied force, suggesting that this motor can change gear. Complementary biochemical kinetic work on yeast dynein mutants hints at the allosteric mechanisms involved.
Subject(s)
Cytoplasm/metabolism , Dyneins/physiology , Models, Biological , Molecular Motor Proteins/physiology , Adenosine Triphosphate/metabolism , Biological Transport , Biomechanical Phenomena , Dyneins/metabolism , Protein Structure, Tertiary/physiologyABSTRACT
An ingenious new experiment used a form of kinesin with one slow head and one fast head to demonstrate definitively that this motor protein moves along a microtubule using alternating left and right steps.
Subject(s)
Kinesins/metabolism , Microtubules/metabolism , Models, Molecular , Molecular Motor Proteins/metabolism , KineticsABSTRACT
Multiphoton excitation was originally projected to improve live cell fluorescence imaging by minimizing photobleaching effects outside the focal plane, yet reports suggest that photobleaching within the focal plane is actually worse than with one photon excitation. We confirm that when imaging enhanced green fluorescent protein, photobleaching is indeed more acute within the multiphoton excitation volume, so that whilst fluorescence increases as predicted with the square of the excitation power, photobleaching rates increase with a higher order relationship. Crucially however, multiphoton excitation also affords unique opportunities for substantial improvements to fluorescence detection. By using a Pockels cell to minimize exposure of the specimen together with multiple nondescanned detectors we show quantitatively that for any particular bleach rate multiphoton excitation produces significantly more signal than one photon excitation confocal microscopy in high resolution Z-axis sectioning of thin samples. Both modifications are readily implemented on a commercial multiphoton microscope system.
Subject(s)
Microscopy, Confocal/methods , Bacteria/cytology , Bacteria/metabolism , Green Fluorescent Proteins , Image Enhancement , Luminescent Proteins/chemistry , Luminescent Proteins/metabolism , Microscopy, Confocal/instrumentation , Photons , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Tubulin/chemistry , Tubulin/metabolismABSTRACT
A recent model suggests that the walking action of kinesin is due to a 13 residue 'fundamental engine' called the neck linker domain, which cyclically zips and unzips to the main part of the heads. New experiments confirm one prediction of the model: that crosslinking the neck linker to the head should block motility.
Subject(s)
Adenosine Triphosphate/metabolism , Kinesins/metabolism , Adenylyl Imidodiphosphate/metabolism , Amino Acid Sequence , Animals , Humans , Kinesins/chemistry , Molecular Sequence Data , Structure-Activity RelationshipABSTRACT
Cryo-electron microscopy and 3D image reconstruction of microtubules saturated with kinesin dimers has shown one head bound to tubulin, the other free. The free head of rat kinesin sits on the top right of the bound head (with the microtubule oriented plus-end upwards) in the presence of 5'-adenylylimido-diphosphate (AMPPNP) and on the top left in nucleotide-free solutions. To understand the relevance of this movement, we investigated other dimeric plus-end-directed motors: Neurospora kinesin (Nkin); Eg5, a slow non-processive kinesin; and a chimera of Ncd heads attached to Nkin necks. In the AMPPNP (ATP-like) state, all dimers have the free head to the top right. In the absence of nucleotide, the free head of an Nkin dimer appears to occupy alternative positions to either side of the bound head. Despite having the Nkin neck, the free head of the chimera was only seen to the top right of the bound head. Eg5 also has the free head mostly to the top right. We suggest that processive movement may require kinesins to move their heads in alternative ways.
Subject(s)
Drosophila Proteins , Kinesins/ultrastructure , Neurospora/chemistry , Recombinant Fusion Proteins/ultrastructure , Xenopus Proteins , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Cryoelectron Microscopy , Dimerization , Image Processing, Computer-Assisted , Kinesins/chemistry , Microtubules/metabolism , Microtubules/ultrastructure , Models, Molecular , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/ultrastructure , Molecular Sequence Data , Movement , Protein Structure, Quaternary , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/chemistry , Sequence AlignmentSubject(s)
Drosophila Proteins , Kinesins/physiology , Molecular Motor Proteins , Kinesins/chemistry , Kinesins/genetics , Movement , MutationABSTRACT
BACKGROUND: Microtubules in interphase Schizosaccharomyces pombe are essential for maintaining the linear growth habit of these cells. The dynamics of assembly and disassembly of these microtubules are so far uncharacterised. RESULTS: Live cell confocal imaging of alpha1 tubulin tagged with enhanced green fluorescent protein revealed longitudinally oriented, dynamically unstable interphase microtubule assemblies (IMAs). The IMAs were uniformly bright along their length apart from a zone of approximately doubly intense fluorescence commonly present close to their centres. The ends of each IMA switched from growth ( approximately 3.0 microm/min) to shrinkage ( approximately 4.5 microm/min) at 1.0 events per minute and from shrinkage to growth at 1.9 events per minute, and the two ends were equivalently dynamic, suggesting equivalent structure. We accordingly propose a symmetrical model for microtubule packing within the IMAs, in which microtubules are plus ends out and overlap close to the equator of the cell. IMAs may contain multiple copies of this motif; if so, then within each IMA end, the microtubule ends must synchronise catastrophe and rescue. When both ends of an IMA lodge in the hemispherical cell ends, the IMAs start to bend under compression and their overall growth rate is inhibited about twofold. Similar microtubule dynamics were observed in cells ranging in size from half to twice normal length. Patterned photobleaching indicated no detectable treadmilling or microtubule sliding during interphase. CONCLUSIONS: The consequence of the mechanisms described is continuous recruitment of microtubule ends to the ends of growing cells, supporting microtubule-based transport into the cell ends and qualitatively accounting for the essential role for microtubules in directing linear cell growth in S. pombe.
Subject(s)
Interphase , Microtubules/metabolism , Schizosaccharomyces/metabolism , Green Fluorescent Proteins , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Mitosis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/genetics , Tubulin/genetics , Tubulin/metabolismABSTRACT
The stepping mechanism of kinesin can be thought of as a programme of conformational changes. We briefly review protein chemical, electron microscopic and transient kinetic evidence for conformational changes, and working from this evidence, outline a model for the mechanism. In the model, both kinesin heads initially trap Mg x ADP. Microtubule binding releases ADP from one head only (the trailing head). Subsequent ATP binding and hydrolysis by the trailing head progressively accelerate attachment of the leading head, by positioning it closer to its next site. Once attached, the leading head releases its ADP and exerts a sustained pull on the trailing head. The rate of closure of the molecular gate which traps ADP on the trailing head governs its detachment rate. A speculative but crucial coordinating feature is that this rate is strain sensitive, slowing down under negative strain and accelerating under positive strain.
Subject(s)
Kinesins/chemistry , Kinesins/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Humans , Kinesins/ultrastructure , Models, Molecular , Protein ConformationABSTRACT
Kinesin is a molecular walking machine with two identical motor heads connected to a coiled-coil tail. Details of the coordination mechanism, which causes kinesin to walk directionally, and the tracking mechanism, which guides each detaching head to its next site on the microtubule, are beginning to emerge.
Subject(s)
Kinesins/genetics , Kinesins/metabolism , Microtubules/physiology , Molecular Motor Proteins/metabolism , Animals , Kinesins/chemistry , Microtubules/metabolism , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/genetics , RatsABSTRACT
Small tension signals that pass between the two linked heads of kinesin allow the motor protein to coordinate its walking action. Two new studies suggest that certain members of the two other major families of motor proteins, the myosins and dyneins, can do the same thing.
Subject(s)
Dyneins/physiology , Kinesins/physiology , Molecular Motor Proteins/physiology , Myosins/physiology , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/physiology , Chlamydomonas/chemistry , Chlamydomonas/physiology , Dyneins/chemistry , Energy Metabolism , Flagella/physiology , Kinesins/chemistry , Macromolecular Substances , Models, Molecular , Molecular Motor Proteins/chemistry , Muscle Contraction/physiology , Myosins/chemistry , Plant Proteins/chemistry , Plant Proteins/physiology , Protein Conformation , Stress, Mechanical , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Adenosine Diphosphate/metabolism , Drosophila Proteins , Kinesins/chemistry , Kinesins/metabolism , Microscopy, Electron/methods , Microtubules/metabolism , Binding Sites , Dimerization , Freezing , Image Processing, Computer-Assisted , Microtubules/ultrastructure , Models, Molecular , Protein Conformation , Tubulin/metabolismABSTRACT
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.
Subject(s)
Kinesins/metabolism , Microscopy, Electron/methods , Microtubules/metabolism , Tubulin/metabolism , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Animals , Apyrase/chemistry , Apyrase/metabolism , Binding Sites/physiology , Cryoelectron Microscopy , Fourier Analysis , Hexokinase/chemistry , Hexokinase/metabolism , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Kinesins/chemistry , Kinesins/ultrastructure , Microscopy, Electron, Scanning , Microtubule Proteins/chemistry , Microtubule Proteins/metabolism , Microtubule Proteins/ultrastructure , Microtubules/chemistry , Microtubules/ultrastructure , Models, Molecular , Molecular Motor Proteins/chemistry , Molecular Motor Proteins/metabolism , Molecular Motor Proteins/ultrastructure , Paclitaxel/chemistry , Protein Binding , Protein Conformation , Protein Structure, Secondary , Swine , Tubulin/chemistry , Tubulin/ultrastructureABSTRACT
To probe for a lever arm action in the kinesin stepping mechanism, we engineered a rodlike extension piece into the tail of rat kinesin at various points close to the head-tail junction and measured its effects on the temperature dependence of velocity in microtubule gliding assays. The insert comprised two contiguous alpha-actinin triple-coil repeats and was predicted to fold into a stiff rodlike module about 11 nm long. The effects of this module were greater the closer it was placed to the head-tail junction. When inserted distal to the head-tail junction, at Asn401 in the dimeric K partial differential401GST, the insert had no effect. When inserted closer to the heads at Val376 into K partial differential376GST, the insert slowed progress below 22 degreesC but accelerated progress to approximately 125% of wild type above 22 degreesC. The most dramatic effect of the synthetic lever occurred when it was inserted very close to the head-neck junction, at Glu340 into the single-headed construct K partial differential340GST. This construct was immotile without the insert, but motile with it, at about 30% of the velocity of the dimeric control. The alpha-actinin module thus confers some gain-of-function when inserted close to the head-neck junction but not when placed distal to it. The data exclude the presence of a lever arm C-terminal to Val376 in the kinesin tail but suggest that a short-throw lever arm may be present, N-terminal to Val376 and contiguous with the head-neck junction at Ala339.
Subject(s)
Kinesins/genetics , Actinin/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , Kinesins/chemistry , Molecular Sequence Data , Mutagenesis, Insertional , Protein Engineering , RatsABSTRACT
Complexes consisting of motor domains of the kinesin-like protein ncd bound to reassembled brain microtubules were visualised using cryoelectron microscopy and helical image reconstruction. Different nucleotide-associated states of a dimeric construct (NDelta295-700) of ncd were analysed to reveal ADP-containing, AMP.PNP-containing and empty (rigor) conformations. In these three states, each thought to mimic a different stage in ATP turnover, the double-headed motors attach to the microtubules by one head only, with the free head tethered in relatively fixed positions. The three structures differ both in the way the attached heads interact with tubulin and in the position of the tethered heads. In the strongly binding rigor and AMP.PNP (ATP-like) states, the attached head makes close contact with both subunits of a tubulin heterodimer. In the weakly bound ADP state, the contact made by the attached head with the monomer closer to the plus end appears to be more loose. Also, in the ATP-like state, the free head tilts nearer to the plus end than in the other two states. The data argue against model mechanisms in which a conformational change in the bound head guides the free head closer to its next binding site; on the contrary, the transition from ADP-filled via rigor to the AMP.PNP (ATP-like) state of the bound head produces a small motion of the free head in the counter-productive direction. However, the observation that the tethered head points towards the minus end, in all three states, is consistent with the idea that the relative arrangement of the heads in a dimer is a major determinant of directionality.
Subject(s)
Drosophila Proteins , Kinesins/chemistry , Microtubules/metabolism , Nucleotides/chemistry , Adenosine Diphosphate/metabolism , Adenylyl Imidodiphosphate/metabolism , Animals , Dimerization , Kinesins/metabolism , Kinesins/ultrastructure , Protein Conformation , Swine , Tubulin/metabolismABSTRACT
We used a battery of proteases to probe the footprint of microtubules on kinesin and ncd, and to search for nucleotide-induced conformational changes in these two oppositely-directed yet homologous molecular motors. Proteolytic cleavage sites were identified by N-terminal microsequencing and electrospray mass spectrometry, and then mapped onto the recently-determined atomic structures of ncd and kinesin. In both kinesin and ncd, microtubule binding shields a set of cleavage sites within or immediately flanking the loops L12, L8 and L11 and, in ncd, the loop L2. Even in the absence of microtubules, exchange of ADP for AMPPNP in the motor active site drives conformational shifts involving these loops. In ncd, a chymotryptic cleavage at Y622 in L12 is protected in the strong binding AMPPNP conformation, but cleaved in the weak binding ADP conformation. In kinesin, a thermolysin cleavage at L154 in L8 is protected in AMPPNP but cleaved in ADP. We speculate that ATP turnover in the active site governs microtubule binding by cyclically retracting or displaying the loops L8 and L12. Curiously, the retracted state of the loops corresponds to microtubule strong binding. Conceivably, nucleotide-dependent display of loops works as a reversible block on strong binding.