Nucleotide-dependent displacement and dynamics of the α-1 helix in kinesin revealed by site-directed spin labeling EPR.
Biochem Biophys Res Commun
; 443(3): 911-6, 2014 Jan 17.
Article
in En
| MEDLINE
| ID: mdl-24361895
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.
Key words
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; Spinspin distance; Structural dynamics; adenosine 5'-(ß,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; ßME
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Spin Labels
/
Kinesins
/
Nucleotides
Type of study:
Prognostic_studies
Language:
En
Journal:
Biochem Biophys Res Commun
Year:
2014
Document type:
Article
Affiliation country: