Nucleotide-dependent displacement and dynamics of the α-1 helix in kinesin revealed by site-directed spin labeling EPR

Biochemical and Biophysical Research Communications
Satoshi YasudaToshiaki Arata

Abstract

In kinesin X-ray crystal structures, the N-terminal region of the α-1 helix is adjacent to the adenine ring of the bound nucleotide, while the C-terminal region of the helix is near the neck-linker (NL). Here, we monitor the displacement of the α-1 helix within a kinesin monomer bound to microtubules (MTs) in the presence or absence of nucleotides using site-directed spin labeling EPR. Kinesin was doubly spin-labeled at the α-1 and α-2 helices, and the resulting EPR spectrum showed dipolar broadening. The inter-helix distance distribution showed that 20% of the spins have a peak characteristic of 1.4-1.7 nm separation, which is similar to what is predicted from the X-ray crystal structure, albeit 80% were beyond the sensitivity limit (>2.5 nm) of the method. Upon MT binding, the fraction of kinesin exhibiting an inter-helix distance of 1.4-1.7 nm in the presence of AMPPNP (a non-hydrolysable ATP analog) and ADP was 20% and 25%, respectively. In the absence of nucleotide, this fraction increased to 40-50%. These nucleotide-induced changes in the fraction of kinesin undergoing displacement of the α-1 helix were found to be related to the fraction in which the NL undocked from the motor core. It is therefore suggested that a shift...Continue Reading

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