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

Highlights: •Dipolar EPR detects the distance between the spin-labeled kinesin α-1 and α-2 helices. •The distance has at least two populations: 1.5 nm (in crystal form: 20%) and >2.5 nm. •The short distance conformer was populated 40% in the apo state with microtubules. •ATP analog or ADP binding caused the 1.5 nm distance to be less populated (∼20%). •The α-1 helix moves closer to the neck-linker (away from α-2) to facilitate docking. -- 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,more » 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 in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.« less
Authors:
;  [1] ;  [2] ;  [3] ;  [2] ;  [1] ;  [1]
  1. Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043 (Japan)
  2. Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577 (Japan)
  3. Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Shido 1314-1, Samuki, Kagawa 769-2193 (Japan)
Publication Date:
OSTI Identifier:
22242281
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 443; Journal Issue: 3; Other Information: Copyright (c) 2013 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ADENOSINE; ADP; ATP; CRYSTAL STRUCTURE; EGTA; ELECTRON MICROSCOPY; ELECTRON SPIN RESONANCE; LABELLING; MICROTUBULES; PIPERAZINES; SPIN