title: Multiple conformations of the nucleotide site of Kinesin family motors in the triphosphate state.
abstract: Identifying conformational changes in kinesin family motors associated with nucleotide and microtubule (MT) binding is essential to determining an atomic-level model for force production and motion by the motors. Using the mobility of nucleotide analog spin probes bound at the active sites of kinesin family motors to monitor conformational changes, we previously demonstrated that, in the ADP state, the open nucleotide site closes upon MT binding [Naber, N., Minehardt, T. J., Rice, S., Chen, X., Grammer, J., Matuska, M., et al. (2003). Closing of the nucleotide pocket of kinesin family motors upon binding to microtubules. Science, 300, 798-801]. We now extend these studies to kinesin-1 (K) and ncd (nonclaret disjunctional protein) motors in ATP and ATP-analog states. Our results reveal structural differences between several triphosphate and transition-state analogs bound to both kinesin and ncd in solution. The spectra of kinesin/ncd in the presence of SLADP•AlFx/BeFx and kinesin, with the mutation E236A (K-E236A; does not hydrolyze ATP) bound to ATP, show an open conformation of the nucleotide pocket similar to that seen in the kinesin/ncd•ADP states. In contrast, the triphosphate analogs K•SLAMPPNP and K-E236A•SLAMPPNP induce a more immobilized component of the electron paramagnetic resonance spectrum, implying closing of the nucleotide site. The MT-bound states of all of the triphosphate analogs reveal two novel spectral components. The equilibrium between these two components is only weakly dependent on temperature. Both components have more restricted mobility than observed in MT-bound diphosphate states. Thus, the closing of the nucleotide pocket when the diphosphate state binds to MTs is amplified in the triphosphate state, perhaps promoting accelerated ATP hydrolysis. Consistent with this idea, molecular dynamics simulations show a good correlation between our spectroscopic data, X-ray crystallography, and the electron microscopy of MT-bound triphosphate-analog states.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, USA.
authors.forename: Nariman
authors.initials: N
authors.lastname: Naber
authors.forename: Adam
authors.initials: A
authors.lastname: Larson
authors.forename: Sarah
authors.initials: S
authors.lastname: Rice
authors.forename: Roger
authors.initials: R
authors.lastname: Cooke
authors.forename: Edward
authors.initials: E
authors.lastname: Pate
mesh_terms: D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D029721:Drosophila Proteins / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000648:ultrastructure; D008868:Microtubule Proteins / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry*; D011122:Polyphosphates / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D029721:Drosophila Proteins; D008868:Microtubule Proteins; D009711:Nucleotides; D011122:Polyphosphates; D016547:Kinesins; C005692:triphosphoric acid