Kinetics of Microtubule Assembly

Cell, 1997

proteins in axonal transport and the isolation of kinesin based on video microscopic assays of movement (Brady, 1985;. From a structural point of view, the major breakthrough was the recent X-ray analysis of the head domains of kinesin and the related motor ncd . The protein has the architecture of an ␣/␤ protein, with a Germany † European Synchrotron Radiation Facility central ␤ sheet sandwiched between three ␣ helices on either side. The structure showed a surprising similarity F-38042 Grenoble France to other nucleotide-binding proteins such as G proteins (e.g., the GTP-binding domains of p21 ras , G␣, or elongation factor Tu) or the ATP-binding domain of myosin. In particular, the homology with myosin sparked expecta-Summary tions that the two motor proteins might exhibit a similar mechanism of motility. There was, however, one missing The dimeric form of the kinesin motor and neck dolink: the lever, an ␣-helical neck domain, was clearly main from rat brain with bound ADP has been solved visible in the myosin structure, but absent from the by X-ray crystallography. The two heads of the dimer kinesin or ncd structures, presumably due to disorder.

The Journal of Cell Biology, 2009

Current Biology, 2006

Molecular Cell, 2006

Kinesin motor proteins release nucleotide upon interaction with microtubules (MTs), then bind and hydrolyze ATP to move along the MT. Although crystal structures of kinesin motors bound to nucleotides have been solved, nucleotide-free structures have not. Here, using cryomicroscopy and three-dimensional (3D) reconstruction, we report the structure of MTs decorated with a Kinesin-14 motor, Kar3, in the nucleotide-free state, as well as with ADP and AMPPNP, with resolution sufficient to show a helices. We find large structural changes in the empty motor, including melting of the switch II helix a4, closure of the nucleotide binding pocket, and changes in the central b sheet reminiscent of those reported for nucleotide-free myosin crystal structures. We propose that the switch II region of the motor controls docking of the Kar3 neck by conformational changes in the central b sheet, similar to myosin, rather than by rotation of the motor domain, as proposed for the Kif1A kinesin motor.

Molecular Cell, 2002

CB227 of each kinesin subclass (Endow, 1999). A third class of kinesins, KinI, has been identified in The Scripps Research Institute 10550 North Torrey Pines Road which the motor core is located Internally within the polypeptide sequence. These proteins exhibit proper-La Jolla, California 92037 2 Cytokinetics ties distinct from other, motile, members of the superfamily, since KinIs have ATP-dependent microtubule de-280 East Grand Avenue, Suite 2 South San Francisco, California 94080 polymerizing activity (Desai et al., 1999). KinIs localize principally to the mitotic spindle of dividing cells and are essential during mitosis (Walczak et al., 1996, Maney et al., 1998). The mitotic spindle has enhanced dynamic Summary

Nature, 2006

Journal of Molecular Biology, 2004

Microtubules are highly dynamic components of the cytoskeleton. They are important for cell movement and they are involved in a variety of transport processes together with motor proteins, such as kinesin. The exact mechanism of these transport processes is not known and so far the focus has been on structural changes within the motor domains, but not within the underlying microtubule structure. Here we investigated the interaction between kinesin and tubulin and our experimental data show that microtubules themselves are changing structure during that process. We studied unstained, vitrified samples of microtubules composed of 15 protofilaments using cryo electron microscopy and helical image analysis. 3D maps of plain microtubules and microtubules decorated with kinesin have been reconstructed to , 17 Å resolution. The ab-tubulin dimer could be identified and, according to our data, aand b-tubulin adopt different conformations in plain microtubules. Significant differences were detected between maps of plain microtubules and microtubule-kinesin complexes. Most pronounced is the continuous axial inter-dimer contact in the microtubulekinesin complex, suggesting stabilized protofilaments along the microtubule axis. It seems, that mainly structural changes within a-tubulin are responsible for this observation. Lateral effects are less pronounced. Following our data, we believe, that microtubules play an active role in intracellular transport processes through modulations of their core structure.

Kinesins form a large and diverse superfamily of proteins involved in numerous important cellular processes. The majority of them are molecular motors moving along microtubules. Conversion of chemical energy into mechanical work is accomplished in a sequence of events involving both biochemical and conformational alternation of the motor structure called the mechanochemical cycle. Different members of the kinesin superfamily can either perform their function in large groups or act as single molecules. Conventional kinesin, a member of the kinesin-1 subfamily, exemplifies the second type of motor which requires tight coordination of the mechanochemical cycle in two identical subunits to accomplish processive movement toward the microtubule plus end. Recent results strongly support an asymmetric handover hand model of ''walking'' for this protein. Conformational strain between two subunits at the stage of the cycle where both heads are attached to the microtubule seems to be a major factor in intersubunit coordination, although molecular and kinetic details of this phenomenon are not yet deciphered. We discuss also current knowledge concerning intersubunit coordination in other kinesin subfamilies. Members of the kinesin-3 class use at least three different mechanisms of movement and can translocate in monomeric or dimeric forms. It is not known to what extent intersubunit coordination takes place in Ncd, a dimeric member of the kinesin-14 subfamily which, unlike conventional kinesin, exercises a power-stroke toward the microtubule minus end. Eg5, a member of the kinesin-5 subfamily is a homotetrameric protein with two kinesin-1-like dimeric halves controlled by their relative orientation on two microtubules. It seems that diversity of subunit organization, quaternary structures and cellular functions in the kinesin superfamily are reflected also by the divergent extent and mechanism of intersubunit coordination during kinesin movement along microtubules.

eLife, 2014

Microtubule-based transport by the kinesin motors, powered by ATP hydrolysis, is essential for a wide range of vital processes in eukaryotes. We obtained insight into this process by developing atomic models for no-nucleotide and ATP states of the monomeric kinesin motor domain on microtubules from cryo-EM reconstructions at 5-6 Å resolution. By comparing these models with existing X-ray structures of ADP-bound kinesin, we infer a mechanistic scheme in which microtubule attachment, mediated by a universally conserved 'linchpin' residue in kinesin (N255), triggers a clamshell opening of the nucleotide cleft and accompanying release of ADP. Binding of ATP re-closes the cleft in a manner that tightly couples to translocation of cargo, via kinesin's 'neck linker' element. These structural transitions are reminiscent of the analogous nucleotide-exchange steps in the myosin and F1-ATPase motors and inform how the two heads of a kinesin dimer 'gate' each other to promote coordinated stepping along microtubules.

The typical function of kinesins is to transport cargo along microtubules. Binding of ATP to microtubule-attached motile kinesins leads to cargo displacement. To better understand the nature of the conformational changes that lead to the power stroke that moves a kinesin's load along a microtubule, we determined the X-ray structure of human kinesin-1 bound to alphabeta-tubulin. The structure defines the mechanism of microtubule-stimulated ATP hydrolysis, which releases the kinesin motor domain from microtubules. It also reveals the structural linkages that connect the ATP nucleotide to the kinesin neck linker, a 15-amino acid segment C terminal to the catalytic core of the motor domain, to result in the power stroke. ATP binding to the microtubule-bound kinesin favors neck-linker docking. This biases the attachment of kinesin's second head in the direction of the movement, thus initiating each of the steps taken.