Kinesin Neck Linker Research Articles

Controlling kinesin motor proteins in nanoengineered systems through a metal‐binding on/off switch

A significant challenge in utilizing kinesin biomolecular motors in integrated nanoscale systems is the ability to regulate motor function in vitro. Here we report a versatile mechanism for reversibly controlling the function of kinesin biomolecular motors independent of the fuel supply (ATP). Our approach relied on inhibiting conformational changes in the neck-linker region of kinesin, a process necessary for microtubule transport. We introduced a chemical switch into the neck-linker of kinesin by genetically engineering three histidine residues to create a Zn(2+)-binding site. Gliding motility of microtubules by the mutant kinesin was successfully inhibited by >/=10 microM Zn(2+), as well as other divalent metals. Motility was successfully restored by removal of Zn(2+) using a number of different chelators. Lastly, we demonstrated the robust and cyclic nature of the switch using sequential Zn(2+)/chelator additions. Overall, this approach to controlling motor function is highly advantageous as it enables control of individual classes of biomolecular motors while maintaining a consistent level of fuel for all motors in a given system or device.

1P-151 ヌクレオチドフリー状態でのキネシンネックリンカーの構造解析(分子モーター(1),第46回日本生物物理学会年会)

1P-151 ヌクレオチドフリー状態でのキネシンネックリンカーの構造解析(分子モーター(1),第46回日本生物物理学会年会)

Thermodynamic Properties of the Kinesin Neck-Region Docking to the Catalytic Core

Kinesin motors move on microtubules by a mechanism that involves a large, ATP-triggered conformational change in which a mechanical element called the neck linker docks onto the catalytic core, making contacts with the core throughout its length. Here, we investigate the thermodynamic properties of this conformational change using electron paramagnetic resonance (EPR) spectroscopy. We placed spin probes at several locations on the human kinesin neck linker and recorded EPR spectra in the presence of microtubules and either 5′-adenylylimidodiphosphate (AMPPNP) or ADP at temperatures of 4–30°C. The free-energy change (Δ G) associated with AMPPNP-induced docking of the neck linker onto the catalytic core is favorable but small, about 3 kJ/mol. In contrast, the favorable enthalpy change (Δ H) and unfavorable entropy change ( TΔ S) are quite large, about 50 kJ/mol. A mutation in the neck linker, V331A/N332A, results in an unfavorable Δ G for AMPPNP-induced zipping of the neck linker onto the core and causes motility defects. These results suggest that the kinesin neck linker folds onto the core from a more unstructured state, thereby paying a large entropic cost and gaining a large amount of enthalpy.

ATP Reorients the Neck Linker of Kinesin in Two Sequential Steps

Recent models of the kinesin mechanochemical cycle provide some conflicting information on how the neck linker contributes to movement. Some spectroscopic approaches suggest a nucleotide-induced order-to-disorder transition in the neck linker. However, cryoelectron microscopic imaging suggests instead that nucleotide alters the orientation of the neck linker when docked on the microtubule surface. Furthermore, since these studies utilized transition state or non-hydrolyzable nucleotide analogs, it is not clear at what point in the ATPase cycle this reorientation of the neck linker occurs. We have addressed this issue by developing a strategy to examine the effect of nucleotide on the orientation of the neck linker based on the technique of fluorescence resonance energy transfer. Transient kinetic studies utilizing this approach support a model in which ATP binding leads to two sequential isomerizations, the second of which reorients the neck linker in relation to the microtubule surface.

Role of the kinesin neck linker and catalytic core in microtubule-based motility.

Kinesin motor proteins execute a variety of intracellular microtubule-based transport functions [1]. Kinesin motor domains contain a catalytic core, which is conserved throughout the kinesin superfamily, followed by a neck region, which is conserved within subfamilies and has been implicated in controlling the direction of motion along a microtubule [2] [3]. Here, we have used mutational analysis to determine the functions of the catalytic core and the approximately 15 amino acid 'neck linker' (a sequence contained within the neck region) of human conventional kinesin. Replacement of the neck linker with a designed random coil resulted in a 200-500-fold decrease in microtubule velocity, although basal and microtubule-stimulated ATPase rates were within threefold of wild-type levels. The catalytic core of kinesin, without any additional kinesin sequence, displayed microtubule-stimulated ATPase activity, nucleotide-dependent microtubule binding, and very slow plus-end-directed motor activity. On the basis of these results, we propose that the catalytic core is sufficient for allosteric regulation of microtubule binding and ATPase activity and that the kinesin neck linker functions as a mechanical amplifier for motion. Given that the neck linker undergoes a nucleotide-dependent conformational change [4], this region might act in an analogous fashion to the myosin converter, which amplifies small conformational changes in the myosin catalytic core [5,6].