Charles V. Sindelar

The beginning of kinesin's force-generating cycle visualized at 9-A resolution.

We have used cryo-electron microscopy of kinesin-decorated microtubules to resolve the structure of the motor protein kinesins crucial nucleotide response elements, switch I and the switch II helix, in kinesins poorly understood nucleotide-free state. Both of the switch elements undergo conformational change relative to the microtubule-free state. The changes in switch I suggest a role for it in “ejecting” adenosine diphosphate when kinesin initially binds to the microtubule. The switch II helix has an N-terminal extension, apparently stabilized by conserved microtubule contacts, implying a microtubule activation mechanism that could convey the state of the bound nucleotide to kinesins putative force-delivering element (the “neck linker”). In deriving this structure, we have adapted an image-processing technique, single-particle reconstruction, for analyzing decorated microtubules. The resulting reconstruction visualizes the asymmetric seam present in native, 13-protofilament microtubules, and this method will provide an avenue to higher-resolution characterization of a variety of microtubule- binding proteins, as well as the microtubule itself.

An atomic-level mechanism for activation of the kinesin molecular motors

Kinesin cytoskeletal motors convert the energy of ATP hydrolysis into stepping movement along microtubules. A partial model of this process has been derived from crystal structures, which show that movement of the motor domain relative to its major microtubule binding element, the switch II helix, is coupled to docking of kinesin’s neck linker element along the motor domain. This docking would displace the cargo in the direction of travel and so contribute to a step. However, the crystal structures do not reveal how ATP binding and hydrolysis govern this series of events. We used cryoelectron microscopy to derive 8–9 Å-resolution maps of four nucleotide states encompassing the microtubule-attached kinetic cycle of a kinesin motor. The exceptionally high quality of these maps allowed us to build in crystallographically determined conformations of kinesin’s key subcomponents, yielding novel arrangements of kinesin’s switch II helix and nucleotide-sensing switch loops. The resulting atomic models reveal a seesaw mechanism in which the switch loops, triggered by ATP binding, propel their side of the motor domain down and thereby elicit docking of the neck linker on the opposite side of the seesaw. Microtubules engage the seesaw mechanism by stabilizing the formation of extra turns at the N terminus of the switch II helix, which then serve as an anchor for the switch loops as they modulate the seesaw angle. These observations explain how microtubules activate kinesin’s ATP-sensing machinery to promote cargo displacement and inform the mechanism of kinesin’s ancestral relative, myosin.

The kinesin-1 motor protein is regulated by a direct interaction of its head and tail

Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiled-coil regions near the enzymatically active heads at the N terminus and the regulatory tails at the C terminus bring these globular elements in proximity and stabilize the folded conformation. However, it has remained a mystery how kinesin-1s microtubule-stimulated ATPase activity is regulated in this folded conformation. Here, we present evidence for a direct interaction between the kinesin-1 head and tail. We photochemically cross-linked heads and tails and produced an 8-Å cryoEM reconstruction of the cross-linked head–tail complex on microtubules. These data demonstrate that a conserved essential regulatory element in the kinesin-1 tail interacts directly and specifically with the enzymatically critical Switch I region of the head. This interaction suggests a mechanism for tail-mediated regulation of the ATPase activity of kinesin-1. In our structure, the tail makes simultaneous contacts with the kinesin-1 head and the microtubule, suggesting the tail may both regulate kinesin-1 in solution and hold it in a paused state with high ADP affinity on microtubules. The interaction of the Switch I region of the kinesin-1 head with the tail is strikingly similar to the interactions of small GTPases with their regulators, indicating that other kinesin motors may share similar regulatory mechanisms.

Template-free 13-protofilament microtubule–MAP assembly visualized at 8 Å resolution

The high-resolution structure of doublecortin-stabilized microtubules provides unprecedented insight into their in vivo architecture.

High-resolution structures of kinesin on microtubules provide a basis for nucleotide-gated force-generation

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 kinesins ‘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. DOI: http://dx.doi.org/10.7554/eLife.04686.001

ATPase Cycle of the Nonmotile Kinesin NOD Allows Microtubule End Tracking and Drives Chromosome Movement

Segregation of nonexchange chromosomes during Drosophila melanogaster meiosis requires the proper function of NOD, a nonmotile kinesin-10. We have determined the X-ray crystal structure of the NOD catalytic domain in the ADP- and AMPPNP-bound states. These structures reveal an alternate conformation of the microtubule binding region as well as a nucleotide-sensitive relay of hydrogen bonds at the active site. Additionally, a cryo-electron microscopy reconstruction of the nucleotide-free microtubule-NOD complex shows an atypical binding orientation. Thermodynamic studies show that NOD binds tightly to microtubules in the nucleotide-free state, yet other nucleotide states, including AMPPNP, are weakened. Our pre-steady-state kinetic analysis demonstrates that NOD interaction with microtubules occurs slowly with weak activation of ADP product release. Upon rapid substrate binding, NOD detaches from the microtubule prior to the rate-limiting step of ATP hydrolysis, which is also atypical for a kinesin. We propose a model for NODs microtubule plus-end tracking that drives chromosome movement.

The Structural Basis of Force Generation by the Mitotic Motor Kinesin-5.

Background: Kinesin-5 motors are important for formation and maintenance of the bipolar mitotic spindle. Results: ATP binding triggers coupled conformational changes of kinesin-5 specific structural elements in the microtubule-bound motor domain. Conclusion: Kinesin-5 mechanochemistry is tuned to its cellular functions. Significance: Subnanometer resolution structure determination of microtubule-bound kinesin-5s and kinetics experiments reveal the molecular basis of their motor properties and of drug inhibition. Kinesin-5 is required for forming the bipolar spindle during mitosis. Its motor domain, which contains nucleotide and microtubule binding sites and mechanical elements to generate force, has evolved distinct properties for its spindle-based functions. In this study, we report subnanometer resolution cryoelectron microscopy reconstructions of microtubule-bound human kinesin-5 before and after nucleotide binding and combine this information with studies of the kinetics of nucleotide-induced neck linker and cover strand movement. These studies reveal coupled, nucleotide-dependent conformational changes that explain many of this motors properties. We find that ATP binding induces a ratchet-like docking of the neck linker and simultaneous, parallel docking of the N-terminal cover strand. Loop L5, the binding site for allosteric inhibitors of kinesin-5, also undergoes a dramatic reorientation when ATP binds, suggesting that it is directly involved in controlling nucleotide binding. Our structures indicate that allosteric inhibitors of human kinesin-5, which are being developed as anti-cancer therapeutics, bind to a motor conformation that occurs in the course of normal function. However, due to evolutionarily defined sequence variations in L5, this conformation is not adopted by invertebrate kinesin-5s, explaining their resistance to drug inhibition. Together, our data reveal the precision with which the molecular mechanism of kinesin-5 motors has evolved for force generation.

Novel septin 9 repeat motifs altered in neuralgic amyotrophy bind and bundle microtubules

Novel septin 9 repeat motifs interact with the acidic C-terminal tails of β-tubulin to promote microtubule bundling and asymmetric neurite growth.

Calcium sensitive ring-like oligomers formed by synaptotagmin

Significance Synaptotagmin-1 is the calcium sensor for synchronous neurotransmitter release. It couples calcium influx to the soluble N-ethylmaleimide–sensitive factor activating protein receptor (SNARE)-catalyzed fusion, but how this coupling happens is unknown. Here, using electron microscopy, we report that the cytosolic domain of synaptotagmin can assemble into ring-like oligomers under calcium-free conditions, and these rings disassemble rapidly upon calcium binding. This process suggests a novel but speculative mechanism to explain calcium coupling, in which the synaptotagmin rings separate the vesicle and plasma membranes and prevent the completion of SNARE complex assembly until the influx of calcium. The synaptic vesicle protein synaptotagmin-1 (SYT) is required to couple calcium influx to the membrane fusion machinery. However, the structural mechanism underlying this process is unclear. Here we report an unexpected circular arrangement (ring) of SYT’s cytosolic domain (C2AB) formed on lipid monolayers in the absence of free calcium ions as revealed by electron microscopy. Rings vary in diameter from 18–43 nm, corresponding to 11–26 molecules of SYT. Continuous stacking of the SYT rings occasionally converts both lipid monolayers and bilayers into protein-coated tubes. Helical reconstruction of the SYT tubes shows that one of the C2 domains (most likely C2B, based on its biochemical properties) interacts with the membrane and is involved in ring formation, and the other C2 domain points radially outward. SYT rings are disrupted rapidly by physiological concentrations of free calcium but not by magnesium. Assuming that calcium-free SYT rings are physiologically relevant, these results suggest a simple and novel mechanism by which SYT regulates neurotransmitter release: The ring acts as a spacer to prevent the completion of the soluble N-ethylmaleimide–sensitive factor activating protein receptor (SNARE) complex assembly, thereby clamping fusion in the absence of calcium. When the ring disassembles in the presence of calcium, fusion proceeds unimpeded.

A vertebrate myosin-I structure reveals unique insights into myosin mechanochemical tuning.

Significance We report the high-resolution structure of a tension-sensing myosin-Ib. We identify a striking unique orientation of structural elements that position the motor’s lever arm. This orientation results in a cavity between the motor and lever arm that holds a 10-residue stretch of N-terminal amino acids, a region that is divergent among myosins. We show the importance of the N-terminal region of myosin in controlling the kinetics and mechanics of the motor. Myosins are molecular motors that power diverse cellular processes, such as rapid organelle transport, muscle contraction, and tension-sensitive anchoring. The structural adaptations in the motor that allow for this functional diversity are not known, due, in part, to the lack of high-resolution structures of highly tension-sensitive myosins. We determined a 2.3-Å resolution structure of apo-myosin-Ib (Myo1b), which is the most tension-sensitive myosin characterized. We identified a striking unique orientation of structural elements that position the motor’s lever arm. This orientation results in a cavity between the motor and lever arm that holds a 10-residue stretch of N-terminal amino acids, a region that is divergent among myosins. Single-molecule and biochemical analyses show that the N terminus plays an important role in stabilizing the post power-stroke conformation of Myo1b and in tuning the rate of the force-sensitive transition. We propose that this region plays a general role in tuning the mechanochemical properties of myosins.