共查询到20条相似文献,搜索用时 15 毫秒
1.
R. J. Stewart Jessica Semerjian Christoph F. Schmidt 《European biophysics journal : EBJ》1998,27(4):353-360
Evidence is presented that the kinesin-related ncd protein is not as processive as kinesin. In low surface density motility
experiments, a dimeric ncd fusion protein behaved mechanistically more similar to non-processive myosins than to the highly
processive kinesin. First, there was a critical microtubule length for motility; only microtubules longer than this critical
length moved in low density ncd surfaces, which suggested that multiple ncd proteins must cooperate to move microtubules in
the surface assay. Under similar conditions, native kinesin demonstrated no critical microtubule length, consistent with the
behavior of a highly processive motor. Second, addition of methylcellulose to decrease microtubule diffusion decreased the
critical microtubule length for motility. Also, the rates of microtubule motility were microtubule length dependent in methylcellulose;
short microtubules, that interacted with fewer ncd proteins, moved more slowly than long microtubules that interacted with
more ncd proteins. In contrast, short microtubules, that interacted with one or a few kinesin proteins, moved on average slightly
faster than long microtubules that interacted with multiple kinesins. We conclude that a degree of processivity as high as
that of kinesin, where a single dimer can move over distances on the order of one micrometer, may not be a general mechanistic
feature of the kinesin superfamily.
Received: 16 September 1997 / Accepted: 4 November 1997 相似文献
2.
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. 相似文献
3.
The kinesin-like ncd protein of Drosophila is a minus end-directed microtubule motor 总被引:36,自引:0,他引:36
The Drosophila ncd gene is required for chromosome segregation during female meiosis. Previous analyses suggested that the ncd gene encoded a protein with sequence similarity to the kinesin motor domain, which suggested that, like kinesin, the ncd protein might be a plus end-directed microtubule motor. Here we describe the expression of ncd protein in E. coli and the initial characterization of the ncd protein's motor properties. The ncd protein is indeed a microtubule motor, but the polarity of movement is minus end directed. The ncd protein also has microtubule bundling activity. These findings limit possible models for the in vivo functions of the ncd protein and suggest that motor proteins with similar sequence can generate movement in opposite directions along a microtubule. 相似文献
4.
Keiko Hirose Jan Lwe Maria Alonso Robert A. Cross Linda A. Amos 《Molecular biology of the cell》1999,10(6):2063-2074
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. 相似文献
5.
We have compared the interaction of ncd (non-claret disjunctional), a kinesin related protein, with microtubules and tubulin heterodimer. Ultracentrifugation experiments revealed that the ncd motor domain, residues 335-700 (ncd335), does not induce tubulin polymerization but stabilizes pre-formed microtubules with a maximum effect at a 1:1 ncd335:tubulin ratio. Ncd335 binding to tubulin or microtubules was estimated by following the change in fluorescence polarization of an exogenous dye attached to Cys670 of ncd335. Ncd335 binding to tubulin (containing GTP or GDP-bound) is characterized by a 2:1 stoichiometry, a higher affinity and an increased sensitivity towards salt, ADP, ATP and AMPPNP, as compared with ncd335 binding to microtubules. Maximum ATPases were 0.06-0.08 sec(-1) and 1.8-2.0 sec(-1) for the ncd335-tubulin and ncd335-microtubules complexes, respectively. Only the polymerized complex is fully functional, suggesting the presence of additional contacts between adjacent protofilaments. Moreover, the data reveal that the oligomeric state of microtubules is a potent regulator for the activity of kinesin related proteins. 相似文献
6.
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. 相似文献
7.
The dimeric structure of the members of the kinesin family of motor proteins determines the individual characteristics of their microtubule-based motility. Crystal structures for ncd and kinesin dimers, which move in opposite directions on microtubules, show possible states of these dimers with ADP bound but give no information about these dimers in solution. Here, low-angle X-ray and neutron scattering were used to investigate their solution structures. Scattering profiles of Drosophila ncd 281-700 (NCD281) and human kinesin 1-420 (hKIN420) were compared with models made from the crystallographically determined structures of NCD281 and rat kinesin 1-379 (rKIN379). From the low-angle region it was found that the radius of gyration (Rg) of NCD281 is 3.60 +/- 0.075 nm, which is in agreement with the crystallography-based model. Scattering by longer ncd constructs (NCD250 and NCD224) is also well fit by the appropriate crystallography-based models. However, the measured Rg of hKIN420, 4.05 +/- 0.075 nm, is significantly smaller than that of the crystallography-based model. In addition, the overall scattering pattern of NCD281 is well fit by the model, but that of hKIN420 is poorly fit. Model calculations indicate that the orientation of the catalytic cores is different from that observed in the rKIN379 crystal structure. Like the crystal structure, the best-fitting models do not show 2-fold symmetry about the neck axis; however, their overall shape more resembles a mushroom than the "T"-like orientation of the catalytic cores found in the crystal structure. The center of mass separations of the catalytic cores in the best-fitting models are 0.7-1 nm smaller than in the crystal structure. 相似文献
8.
The dimeric structure of kinesin superfamily proteins plays an important role in their motile functions and characteristics. In this study, the coiled-coil-forming property of the stalk region (192-346) of Drosophila ncd, a C-terminal kinesin motor protein, was investigated by synthesizing various peptide fragments. The alpha helicity of a set of 46-residue peptides spanning the stalk region appeared too low to form a coiled-coil dimer, probably because of insufficient continuity of the hydrophobic residues at (a and d) core positions in amphipathic heptad repeats. On the other hand, several peptides with leucine residues introduced at core positions or with extensional sequences with high alpha helicity had an advantage in coiled-coil formation. When we analyzed the thermal and urea-induced unfolding of these dimeric peptides, we identified four domains having a relatively high potential to form coiled coils. Among them, three domains on the C-terminal side of the stalk region, i.e., (252-272), (276-330), and (336-346), were in the same heptad frame, although these potential coiled-coil domains were not self-sustaining individually. This is in sharp contrast to the fragment of human kinesin, (332-369), which has an extremely high tendency toward coiled-coil formation. One of the possible triggers for coiled-coil formation of the ncd stalk region may be the interaction between the motor domain and the C-terminal part of the stalk as previously revealed by X-ray crystallography. The residues, S331 and R335, seem to act as a breaking point for alpha-helix continuity. This would make the region (336-346), as the head-stalk joint, more flexible such as seen with a plus-end-directed kinesin, if this region had no interaction with the motor domain. These characteristic differences between ncd and kinesin suggest that the nonlocally sustained coiled coil of ncd is one of the factors important for minus-end-directed motility. 相似文献
9.
Wendt TG Volkmann N Skiniotis G Goldie KN Müller J Mandelkow E Hoenger A 《The EMBO journal》2002,21(22):5969-5978
We used cryo-electron microscopy and image reconstruction to investigate the structure and microtubule-binding configurations of dimeric non-claret disjunctional (ncd) motor domains under various nucleotide conditions, and applied molecular docking using ncd's dimeric X-ray structure to generate a mechanistic model for force transduction. To visualize the alpha-helical coiled-coil neck better, we engineered an SH3 domain to the N-terminal end of our ncd construct (296-700). Ncd exhibits strikingly different nucleotide-dependent three-dimensional conformations and microtubule-binding patterns from those of conventional kinesin. In the absence of nucleotide, the neck adapts a configuration close to that found in the X-ray structure with stable interactions between the neck and motor core domain. Minus-end-directed movement is based mainly on two key events: (i) the stable neck-core interactions in ncd generate a binding geometry between motor and microtubule which places the motor ahead of its cargo in the minus-end direction; and (ii) after the uptake of ATP, the two heads rearrange their position relative to each other in a way that promotes a swing of the neck in the minus-end direction. 相似文献
10.
Proteolytic mapping of kinesin/ncd-microtubule interface: nucleotide-dependent conformational changes in the loops L8 and L12. 总被引:4,自引:1,他引:3
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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. 相似文献
11.
Kozielski F De Bonis S Burmeister WP Cohen-Addad C Wade RH 《Structure (London, England : 1993)》1999,7(11):1407-1416
BACKGROUND: The kinesin superfamily of microtubule-associated motor proteins are important for intracellular transport and for cell division in eukaryotes. Conventional kinesins have the motor domain at the N terminus of the heavy chain and move towards the plus end of microtubules. The ncd protein is necessary for chromosome segregation in meiosis. It belongs to a subfamily of kinesins that have the motor domain at the C terminus and move towards the minus end of microtubules. RESULTS: The crystal structure of dimeric ncd has been obtained at 2.9 A resolution from crystals with the C222(1) space group, with two independent dimers per asymmetric unit. The motor domains in these dimers are not related by crystallographic symmetry and the two ncd dimers have significantly different conformations. An alpha-helical coiled coil connects, and interacts with, the motor domains. CONCLUSIONS: The ncd protein has a very compact structure, largely due to extended interactions of the coiled coil with the head domains. Despite this, we find that the overall conformation of the ncd dimer can be rotated by as much as 10 degrees away from that of the twofold-symmetric archetypal ncd. The crystal structures of conventional kinesin and of ncd suggest a structural rationale for the reversal of the direction of movement in chimeric kinesins. 相似文献
12.
Sablin EP 《Current opinion in cell biology》2000,12(1):35-41
Atomic resolution three-dimensional structures of two oppositely directed kinesin motors - conventional kinesin and non-claret disjunctional (ncd) protein - are now available in their functional dimeric form. A detailed model of the microtubule has also been recently obtained by docking the 3.7 A structure of tubulin into a 20 A map of the microtubule. Recent structural studies of kinesin motors and their microtubule tracks are contributing to our current understanding of kinesin motor mechanisms. 相似文献
13.
Ncd is a microtubule minus-end directed motor of the kinesin superfamily. Previously it has been shown that ncd and kinesin motor domains share the same major binding site on microtubules. Here we report a three-dimensional EM reconstruction of negatively stained two-dimensional Zn-induced tubulin crystal sheets (Zn-sheets) decorated with the ncd motor domain at a resolution of 16 A. This work has revealed a second specific binding site for the ncd motor domain. The motor binding site on the tubulin Zn-sheets spans both alpha and beta tubulin subunits. This binding site is located at a position different from the previously identified ncd binding site on microtubules and may play a role in motor function. 相似文献
14.
Hoenger A Thormählen M Diaz-Avalos R Doerhoefer M Goldie KN Müller J Mandelkow E 《Journal of molecular biology》2000,297(5):1087-1103
The interactions of monomeric and dimeric kinesin and ncd constructs with microtubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structure of dimeric ncd when bound to a tubulin dimer showing one head attached directly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations. Here we reinvestigated the microtubule binding patterns of dimeric kinesins by cryo-EM and digital 3D reconstruction under different nucleotide conditions and different motor:tubulin ratios, and determined the molecular mass of motor-tubulin complexes by STEM. Both methods revealed complementary results. We found that the ratio of bound kinesin motor-heads to alphabeta-tubulin dimers was never reaching above 1.5 irrespective of the initial mixing ratios. It appears that each kinesin dimer occupies two microtubule-binding sites, provided that there is a free one nearby. Thus the appearances of different image reconstructions can be explained by non-specific excess binding of motor heads. Consequently, the use of different apparent density distributions for docking the X-ray structures onto the microtubule surface leads to different and mutually exclusive models. We propose that in conditions of stoichiometric binding the two heads of a kinesin dimer separate and bind to different tubulin subunits. This is in contrast to ncd where the two heads remain tightly attached on the microtubule surface. Using dimeric kinesin molecules crosslinked in their neck domain we also found that they stabilize protofilaments axially, but not laterally, which is a strong indication that the two heads of the dimers bind along one protofilament, rather than laterally bridging two protofilaments. A molecular walking model based on these results summarizes our conclusions and illustrates the implications of symmetry for such models. 相似文献
15.
The shapes of the motor domains of two oppositely directed microtubule motors, ncd and kinesin: a neutron scattering study. 总被引:1,自引:0,他引:1
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The shapes of the motor domains of kinesin and ncd, which move in opposite directions along microtubules, have been investigated. Using proteins expressed in Escherichia coli, it was found that at high salt (> 200 mM) Drosophila ncd motor domain (R335-K700) and human kinesin motor domain (M1-E349) were both sufficiently monomeric to allow an accurate determination of their radii of gyration (Rg) and their molecular weights. The measured Rg values of the ncd and kinesin motor domains in D2O were 2.06 +/- 0.06 and 2.05 +/- 0.04 nm, respectively, and the molecular weights were consistent with those computed from the amino acid compositions. Fitting of the scattering curves to approximately 3.5 nm resolution showed that the ncd and kinesin motor domains can be described adequately by triaxial ellipsoids having half-axes of 1.42 +/- 0.38, 2.24 +/- 0.44, and 3.65 +/- 0.22 nm, and half-axes of 1.52 +/- 0.23, 2.00 +/- 0.25, and 3.73 +/- 0.10 nm, respectively. Both motor domains are described adequately as somewhat flattened prolate ellipsoids with a maximum dimension of approximately 7.5 nm. Thus, it appears that the overall shapes of these motor domains are not the major determinants of the directionality of their movement along microtubules. 相似文献
16.
Nucleotide-dependent movements of the kinesin motor domain predicted by simulated annealing.
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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. 相似文献
17.
Formation of membrane networks in vitro by kinesin-driven microtubule movement 总被引:19,自引:15,他引:4
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《The Journal of cell biology》1988,107(6):2233-2241
Certain intracellular organelles such as the endoplasmic reticulum (Terasaki, M., L. B. Chen, and K. Fujiwara. 1986. J. Cell Biol. 103:1557-1568) and lysosomes (Swanson, J., A. Bushnell, and S. C. Silverstein. Proc. Natl. Acad. Sci. USA. 84:1921-1925) form tubular networks that are closely aligned with microtubules. Here we describe the formation of polygonal networks composed of interconnected membrane tubules that occurs when a preparation of microtubule affinity-purified squid kinesin is combined with microtubules and ATP on a glass surface. The membrane, which is a minor contaminant in the microtubule affinity- purified kinesin preparation, binds to microtubules translocating along kinesin-coated glass surfaces. Force exerted by kinesin upon the microtubule is transmitted to the membrane and a tubular extension of the membrane is produced. As the membrane tubule elongates, membrane tension exerts an opposing force upon the translocating microtubule that can alter its direction of movement by dissociating or partially dissociating the microtubule from the kinesin-coated surface. Membrane tubules that come in contact appear to fuse with one another, and thus give rise to two-dimensional polygonal networks of tubules that have similar features to endoplasmic reticulum networks in cells. Artificial liposomes composed of dimyristoylphosphatidylcholine and yolk phosphatidylglycerol also form stable tubular structures when subjected to shear forces, but do not interact with microtubules or form polygonal networks, suggesting that such phenomena may require membrane- associated proteins. These findings indicate that kinesin generates sufficient force to form tubular membrane extensions in vitro and suggest that this microtubule-based motility protein may also be responsible for creating tubular membrane networks within cells. 相似文献
18.
Ncd is a microtubule minus end-directed motor protein from Drosophila, a member of the kinesin-14 family, and an essential protein in mitosis and meiosis. Full-length ncd exists as a dimer via the formation of an alpha-helical coiled coil in its central stalk domain (P192-R346), which is thought to be one of the key regions for its motility. In our previous studies, however, none of the various synthetic polypeptide fragments (up to 46 residues) from the stalk domain formed a coiled coil. Herein, we have investigated the structural properties of the full-length ncd stalk domain using recombinant polypeptides together with shorter segments. These new fragments did form coiled coils as verified by far-UV circular dichroism (CD) spectroscopy and analytical ultracentrifugation, suggesting that a certain length of polypeptide would be required for dimer formation. Moreover, deletion mapping revealed that the cooperativity among the neighboring subdomains in the stalk domain is required for formation of the coiled coil. Interestingly, the intact stalk domain segments showed three-state transition in thermal unfolding measurements with CD, indicating the presence of two regions: (i) a coiled-coil region (P227-R306) that exhibits reversible denaturation at a lower temperature (20-30 degrees C) and (ii) a more rigid coiled-coil region (T307-E334) that exhibits irreversible denaturation at a high temperature (ca. 60 degrees C). These results imply that the N-terminal region of the stalk domain might be able to adopt both a coiled-coil conformation and a dissociated one, which might be relevant to the functions of ncd. 相似文献
19.
Microtubules and associated motor proteins such as kinesin are envisioned for applications such as bioseparation and molecular sorting to powering hybrid synthetic mechanical devices. One of the challenges in realizing such systems is retaining motor functionality on device surfaces. Kinesin motors adsorbed onto glass surfaces lose their functionality or ability to interact with microtubules if not adsorbed with other supporting proteins. Casein, a milk protein, is commonly used in microtubule motility assays to preserve kinesin functionality. However, the mechanism responsible for this preservation of motor function is unknown. To study casein and kinesin interaction, a series of microtubule motility assays were performed where whole milk casein, or its αs1 and αs2, β or κ subunits, were introduced or omitted at various steps of the motility assay. In addition, a series of epifluorescence and total internal reflection microscopy (TIRF) experiments were conducted where fluorescently labeled casein was introduced at various steps of the motility assay to assess casein-casein and casein-glass binding dynamics. From these experiments it is concluded that casein forms a bi-layer which supports the operation of kinesin. The first tightly bound layer of casein mainly performs the function of anchoring the kinesin while the second more loosely bound layer of casein positions the head domain of the kinesin to more optimally interact with microtubules. Studies on individual casein subunits indicate that β casein was most effective in supporting kinesin functionality while κ casein was found to be least effective. 相似文献
20.
The motor domain of the kinesin homolog ncd has been crystallized in the presence of MgATP by the vapor diffusion method using polyethylene glycol as the precipitant. The crystals belong to the orthorhombic space group I222 with unit cell dimensions a = 127.1 Å, b = 122.3 Å, c = 68.0 Å, and there is one ncd molecule per asymmetric unit. The crystals diffract X-ray to at least 2.3 Å and are appropriate for high-resolution structure determination. © 1995 Wiley-Liss, Inc. 相似文献