Nucleotide-dependent displacement and dynamics of the 伪-1 helix in kinesin revealed by site-directed spin labeling EPR
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- 作者：Satoshi Yasuda ; Takanori Yanagi ; Masafumi D. Yamada ; Shoji Ueki ; Shinsaku Maruta ; Akio Inoue ; Toshiaki Arata
- 关键词：AMPPNP ; adenosine 5&prime ; -(尾 ; 纬-imido)triphosphate ; 尾ME ; 2-mercaptoethanol ; CW ; continuous wave ; EGTA ; ethylene glycol-bis(l-aminoethyl ether)-N ; N ; N&prime ; N&prime ; -tetraacetic acid ; EM ; electron microscopy ; MSL ; 4-maleimido-2 ; 2 ; 6 ; 6-tetramethyl-1-piperidinoxy ; NN ; no nucleotide ; MT ; microtubule ; PIPES ; piperazine-1 ; 4-bis(2-ethanesulfonic acid) ; SDSL ; site-directed spin labeling
- 刊名：Biochemical and Biophysical Research Communications
- 出版年：17 January, 2014
- 年：2014
- 卷：443
- 期：3
- 页码：911-916
- 全文大小：1114 K
文摘
In kinesin X-ray crystal structures, the N-terminal region of the 伪-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 伪-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 伪-1 and 伪-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 伪-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 伪-1 helix conformational equilibrium occurs upon nucleotide binding and release, and this shift controls NL docking onto the motor core.