EPR spectroscopy shows a microtubule-dependent conformational change in the kinesin switch 1 domain.
We have used site-directed spin-labeling and electron paramagnetic resonance spectroscopy to monitor a conformational change at the nucleotide site of kinesin. Cys-lite kinesin (K349 monomer) with the mutation S188C was spin labeled with MSL or MTSL. This residue is at the junction between the switch 1 region (which is a structure known to be sensitive to bound nucleotide in the G-proteins) and the alpha3-helix, adjacent to the nucleotide site. The spectra showed two or more components of mobility, which were independent of nucleotide in the absence of microtubules (MTs). The spectra of both labels showed a change of mobility upon binding to MTs. A more mobile spectral component became enhanced for all triphosphate analogs examined, AMPPNP, ADP.AlFx, or ADP.BeFx, in the presence of MTs, although the magnitude of the new component and the degree of mobility varied with nucleotide analog. The ADP state showed a much-reduced spectral change with a small shift to the more immobilized component in the presence of MTs. For kinesin.ADP.MT, a van't Hoff plot gave DeltaH degrees = -96 kJ/mol implying that the conformational change was extensive. We conclude there is a conformational change in the switch 1-alpha3-helix domain when kinesin binds to MTs.
Naber
Nariman
N
Department of Biochemistry and Biophysics, Department of Cellular and Molecular Pharmacology, and Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA.
Rice
Sarah
S
Matuska
Marija
M
Vale
Ronald D
RD
Cooke
Roger
R
Pate
Edward
E
D000227:Adenine Nucleotides / Q000737:chemistry; D001665:Binding Sites; D003553:Cystine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D016547:Kinesins / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry*; D020409:Molecular Motor Proteins / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013113:Spin Labels; D013696:Temperature; D014404:Tubulin / Q000737:chemistry
D000227:Adenine Nucleotides; D020409:Molecular Motor Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; D013113:Spin Labels; D014404:Tubulin; D003553:Cystine; D016547:Kinesins