title: Nucleotide-dependent displacement and dynamics of the α-1 helix in kinesin revealed by site-directed spin labeling EPR.
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 in the α-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yasuda
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Yanagi
authors.affiliation: Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Masafumi D
authors.initials: MD
authors.lastname: Yamada
authors.affiliation: Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Shido 1314-1, Samuki, Kagawa 769-2193, Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Ueki
authors.affiliation: Division of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Maruta
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Inoue
authors.affiliation: Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan. Electronic address: arata@bio.sci.osaka-u.ac.jp.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Arata
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry; D009711:Nucleotides / Q000378:metabolism*; D017433:Protein Structure, Secondary; D012399:Rotation; D013113:Spin Labels
chemical_list: D050505:Mutant Proteins; D009711:Nucleotides; D013113:Spin Labels; D000251:Adenosine Triphosphatases; D016547:Kinesins
keywords: 2-mercaptoethanol; 4-maleimido-2,2,6,6-tetramethyl-1-piperidinoxy; AMPPNP; CW; Conventional kinesin; Dipolar EPR; EGTA; EM; Electron paramagnetic resonance; Kinesin-1; MSL; MT; Molecular motor; NN; Neck-linker; PIPES; SDSL; Spin labeling; Spin–spin distance; Structural dynamics; adenosine 5′-(β,γ-imido)triphosphate; continuous wave; electron microscopy; ethylene glycol-bis(l-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; microtubule; no nucleotide; piperazine-1,4-bis(2-ethanesulfonic acid); site-directed spin labeling; βME