Jared C. Cochran

Modulation of kinesin binding by the C-termini of tubulin.

The flexible tubulin C‐terminal tails (CTTs) have recently been implicated in the walking mechanism of dynein and kinesin. To address their role in the case of conventional kinesin, we examined the structure of kinesin–microtubule (MT) complexes before and after CTT cleavage by subtilisin. Our results show that the CTTs directly modulate the motor–tubulin interface and the binding properties of motors. CTT cleavage increases motor binding stability, and kinesin appears to adopt a binding conformation close to the nucleotide‐free configuration under most nucleotide conditions. Moreover, C‐terminal cleavage results in trapping a transient motor–ADP–MT intermediate. Using SH3‐tagged dimeric and monomeric constructs, we could also show that the position of the kinesin neck is not affected by the C‐terminal segments of tubulin. Overall, our study reveals that the tubulin C‐termini define the stability of the MT–kinesin complex in a nucleotide‐dependent manner, and highlights the involvement of tubulin in the regulation of weak and strong kinesin binding states.

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 Loop 5 Element Structurally and Kinetically Coordinates Dimers of the Human Kinesin-5, Eg5

Eg5 is a homotetrameric kinesin-5 motor protein that generates outward force on the overlapping, antiparallel microtubules (MTs) of the mitotic spindle. Upon binding an MT, an Eg5 dimer releases one ADP molecule, undergoes a slow (∼0.5 s(-1)) isomerization, and finally releases a second ADP, adopting a tightly MT-bound, nucleotide-free (APO) conformation. This conformation precedes ATP binding and stepping. Here, we use mutagenesis, steady-state and pre-steady-state kinetics, motility assays, and electron paramagnetic resonance spectroscopy to examine Eg5 monomers and dimers as they bind MTs and initiate stepping. We demonstrate that a critical element of Eg5, loop 5 (L5), accelerates ADP release during the initial MT-binding event. Furthermore, our electron paramagnetic resonance data show that L5 mediates the slow isomerization by preventing Eg5 dimer heads from binding the MT until they release ADP. Finally, we find that Eg5 having a seven-residue deletion within L5 can still hydrolyze ATP and move along MTs, suggesting that L5 is not required to accelerate subsequent steps of the motor along the MT. Taken together, these properties of L5 explain the kinetic effects of L5-directed inhibition on Eg5 activity and may direct further interventions targeting Eg5 activity.

A metal switch for controlling the activity of molecular motor proteins

Kinesins are molecular motors that require a divalent metal ion (for example, Mg2+) to convert the energy of ATP hydrolysis into directed force production along microtubules. Here we present the crystal structure of a recombinant kinesin motor domain bound to Mn2+ and ADP and report on a serine-to-cysteine substitution in the switch 1 motif of kinesin that allows its ATP hydrolysis activity to be controlled by adjusting the ratio of Mn2+ to Mg2+. This mutant kinesin binds ATP similarly in the presence of either metal ion, but its ATP hydrolysis activity is greatly diminished in the presence of Mg2+. In human kinesin-1 and kinesin-5 as well as Drosophila melanogaster kinesin-10 and kinesin-14, this defect is rescued by Mn2+, providing a way to control both the enzymatic activity and force-generating ability of these nanomachines.

Modulation of the Kinesin ATPase Cycle by Neck Linker Docking and Microtubule Binding

Kinesin motor proteins use an ATP hydrolysis cycle to perform various functions in eukaryotic cells. Many questions remain about how the kinesin mechanochemical ATPase cycle is fine-tuned for specific work outputs. In this study, we use isothermal titration calorimetry and stopped-flow fluorometry to determine and analyze the thermodynamics of the human kinesin-5 (Eg5/KSP) ATPase cycle. In the absence of microtubules, the binding interactions of kinesin-5 with both ADP product and ATP substrate involve significant enthalpic gains coupled to smaller entropic penalties. However, when the wild-type enzyme is titrated with a non-hydrolyzable ATP analog or the enzyme is mutated such that it is able to bind but not hydrolyze ATP, substrate binding is 10-fold weaker than ADP binding because of a greater entropic penalty due to the structural rearrangements of switch 1, switch 2, and loop L5 on ATP binding. We propose that these rearrangements are reversed upon ATP hydrolysis and phosphate release. In addition, experiments on a truncated kinesin-5 construct reveal that upon nucleotide binding, both the N-terminal cover strand and the neck linker interact to modulate kinesin-5 nucleotide affinity. Moreover, interactions with microtubules significantly weaken the affinity of kinesin-5 for ADP without altering the affinity of the enzyme for ATP in the absence of ATP hydrolysis. Together, these results define the energy landscape of a kinesin ATPase cycle in the absence and presence of microtubules and shed light on the role of molecular motor mechanochemistry in cellular microtubule dynamics.

Recombinant production of TEV cleaved human parathyroid hormone.

The parathyroid hormone, PTH, is responsible for calcium and phosphate ion homeostasis in the body. The first 34 amino acids of the peptide maintain the biological activity of the hormone and is currently marketed for calcium imbalance disorders. Although several methods for the production of recombinant PTH(1‐34) have been reported, most involve the use of cleavage conditions that result in a modified peptide or unfavorable side products. Herein, we detail the recombinant production of 15N‐enriched human parathyroid hormone, 15N PTH(1‐34), generated via a plasmid vector that gives reasonable yield, low‐cost protease cleavage (leaving the native N‐terminal serine in its amino form), and purification by affinity and size exclusion chromatography. We characterize the product by multidimensional, heteronuclear NMR, circular dichroism, and LC/MS. Copyright

Kinesin Motors: No Strain, No Gain

The processive movement of the dimeric motor protein kinesin 1 along microtubules requires communication between the two motor domains. Yildiz et al. (2008) now show that tension between the motor domains not only is necessary for normal processivity but also may be sufficient for motor motility under some conditions.

A molecular motor finds its track

The detailed mechanism by which the molecular motors kinesin and myosin travel along their respective protein tracks as they generate force during motile processes is still poorly understood. In a recent breakthrough, a crystal structure of kinesin in complex with tubulin illuminates the atomic-level details of a motor-track interaction, answering many questions yet leaving a number of mysteries unresolved.

Protein engineering of the N-terminus of NEMO: structure stabilization and rescue of IKKβ binding.

NEMO is a scaffolding protein that, together with the catalytic subunits IKKα and IKKβ, plays an essential role in the formation of the IKK complex and in the activation of the canonical NF-κB pathway. Rational drug design targeting the IKK-binding site on NEMO would benefit from structural insight, but to date, the determination of the structure of unliganded NEMO has been hindered by protein size and conformational heterogeneity. Here we show how the utilization of a homodimeric coiled-coil adaptor sequence stabilizes the minimal IKK-binding domain NEMO(44–111) and furthers our understanding of the structural requirements for IKK binding. The engineered constructs incorporating the coiled coil at the N-terminus, C-terminus, or both ends of NEMO(44–111) present high thermal stability and cooperative melting and, most importantly, restore IKKβ binding affinity. We examined the consequences of structural content and stability by circular dichoism and nuclear magnetic resonance (NMR) and measured the binding affinity of each construct for IKKβ(701–745) in a fluorescence anisotropy binding assay, allowing us to correlate structural characteristics and stability to binding affinity. Our results provide a method for engineering short stable NEMO constructs to be suitable for structural characterization by NMR or X-ray crystallography. Meanwhile, the rescuing of the binding affinity implies that a preordered IKK-binding region of NEMO is compatible with IKK binding, and the conformational heterogeneity observed in NEMO(44–111) may be an artifact of the truncation.