A neuropathy-associated kinesin KIF1A mutation hyper-stabilizes the motor-neck interaction during the ATPase cycle.

The mechanochemical coupling of ATPase hydrolysis and conformational dynamics in kinesin motors facilitates intramolecular interaction cycles between the kinesin motor and neck domains, which are essential for microtubule-based motility. Here, we characterized a charge-inverting KIF1A-E239K mutant that we identified in a family with axonal-type Charcot-Marie-Tooth disease and also in 24 cases in human neuropathies including spastic paraplegia and hereditary sensory and autonomic neuropathy. We show that Glu239 in the ß7 strand is a key residue of the motor domain that regulates the motor-neck interaction. Expression of the KIF1A-E239K mutation has decreased ability to complement Kif1a+/- neurons, and significantly decreases ATPase activity and microtubule gliding velocity. X-ray crystallography shows that this mutation causes an excess positive charge on ß7, which may electrostatically interact with a negative charge on the neck. Quantitative mass spectrometric analysis supports that the mutation hyper-stabilizes the motor-neck interaction at the late ATP hydrolysis stage. Thus, the negative charge of Glu239 dynamically regulates the kinesin motor-neck interaction, promoting release of the neck from the motor domain upon ATP hydrolysis.

Kinesin-binding-triggered conformation switching of microtubules contributes to polarized transport.

Kinesin-1, the founding member of the kinesin superfamily of proteins, is known to use only a subset of microtubules for transport in living cells. This biased use of microtubules is proposed as the guidance cue for polarized transport in neurons, but the underlying mechanisms are still poorly understood. Here, we report that kinesin-1 binding changes the microtubule lattice and promotes further kinesin-1 binding. This high-affinity state requires the binding of kinesin-1 in the nucleotide-free state. Microtubules return to the initial low-affinity state by washing out the binding kinesin-1 or by the binding of non-hydrolyzable ATP analogue AMPPNP to kinesin-1. X-ray fiber diffraction, fluorescence speckle microscopy, and second-harmonic generation microscopy, as well as cryo-EM, collectively demonstrated that the binding of nucleotide-free kinesin-1 to GDP microtubules changes the conformation of the GDP microtubule to a conformation resembling the GTP microtubule.

X-ray and Cryo-EM structures reveal mutual conformational changes of Kinesin and GTP-state microtubules upon binding.

The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state microtubules over GDP-state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide-free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide-free KIF5C and the cryo-electron microscopic structure of nucleotide-free KIF5C complexed with the GTP-state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP-state microtubule and KIF5C. KIF5C acquires the 'rigor conformation', where mobile switches I and II are stabilized through L11 and the initial portion of the neck-linker, facilitating effective ADP release and the weak-to-strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP-state microtubule in a similar manner to GDP-taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP-state microtubules.

Oxidative stress-mediated bimodal regulation of polymorphonuclear leukocyte spreading by polyphenolic compounds.

Pyrogallol-bearing polyphenolic compounds induce spreading of polymorphonuclear leukocytes (PMNL), although their optimal concentrations for induction of spreading are quite different (2000, 200, and 2 µM for pyrogallol, (-)-epigallocatechin gallate (EGCG), and tannic acid (TA), respectively), and TA tends to inhibit spreading at higher concentrations. In this study, we examined the involvement of oxidative stress in the regulation of PMNL spreading by these compounds. All three compounds in solution generated H(2)O(2) to a similar extent. Adsorption of the polyphenols to cell surfaces and their accumulation within cells were assessed by detection of the H(2)O(2) precursor O(2)(-) produced by the compounds through reduction of cytochrome c and p-nitro-blue tetrazolium, respectively. TA showed the highest degree of adsorption. EGCG adhered only to PMNL pre-fixed by paraformaldehyde, whereas pyrogallol did not adhere. None of the compounds caused intracellular O(2)(-) generation. A non-pyrogallic compound, 1,2,4-benzenetriol (BT), also produced H(2)O(2); it had no stimulatory effect on PMNL spreading, but inhibited spreading induced by other stimuli. BT did not adhere to PMNL but accumulated within them, and generated O(2)(-) in the presence of glycine. Thiol antioxidants abrogated all of the above spreading-regulatory effects of the polyphenolic compounds. We conclude that H(2)O(2)-generating polyphenols bimodally regulate the spreading of PMNL by subjecting them to oxidative stress. The ability of polyphenol to adhere to, or accumulate within, PMNL may govern the nature of the oxidative stress and determine the optimal concentration of each compound for induction of spreading, as well as whether spreading is promoted or inhibited.