Single-molecule observations of neck linker conformational changes in the kinesin motor protein

Kinesin-1 is a dimeric motor protein that moves cargo processively along microtubules. Kinesin motility has been proposed to be driven by the coordinated forward extension of the neck linker (a ∼12-residue peptide) in one motor domain and the rearward positioning of the neck linker in the partner motor domain. To test this model, we have introduced fluorescent dyes selectively into one subunit of the kinesin dimer and performed 'half-molecule' fluorescence resonance energy transfer to measure conformational changes of the neck linker. We show that when kinesin binds with both heads to the microtubule, the neck linkers in the rear and forward heads extend forward and backward, respectively. During ATP-driven motility, the neck linkers switch between these conformational states. These results support the notion that neck linker movements accompany the 'hand-over-hand' motion of the two motor domains.

[1]  E. Mandelkow,et al.  The Crystal Structure of Dimeric Kinesin and Implications for Microtubule-Dependent Motility , 1997, Cell.

[2]  W. Schief,et al.  Conformational changes during kinesin motility. , 2001, Current opinion in cell biology.

[3]  R. Fletterick,et al.  Coordination between Motor Domains in Processive Kinesins* , 2004, Journal of Biological Chemistry.

[4]  Yale E. Goldman,et al.  Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization , 2003, Nature.

[5]  R. Cross,et al.  Kinesin's moonwalk. , 2006, Current opinion in cell biology.

[6]  Zygmunt Gryczynski,et al.  A FRET-Based Sensor Reveals Large ATP Hydrolysis–Induced Conformational Changes and Three Distinct States of the Molecular Motor Myosin , 2000, Cell.

[7]  R. Cross,et al.  Mechanics of the kinesin step , 2005, Nature.

[8]  R Vale,et al.  Thermodynamic properties of the kinesin neck-region docking to the catalytic core. , 2003, Biophysical journal.

[9]  Ronald D. Vale,et al.  Controlling Kinesin by Reversible Disulfide Cross-Linking , 2000, The Journal of cell biology.

[10]  E. Mandelkow,et al.  Nucleotide‐induced conformations in the neck region of dimeric kinesin , 2003, The EMBO journal.

[11]  R. Vale,et al.  Kinesin Walks Hand-Over-Hand , 2004, Science.

[12]  Nobutaka Hirokawa,et al.  Molecular motors and mechanisms of directional transport in neurons , 2005, Nature Reviews Neuroscience.

[13]  Roger Cooke,et al.  A structural change in the kinesin motor protein that drives motility , 1999, Nature.

[14]  D. Hackney,et al.  Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  S. Rosenfeld,et al.  ATP Reorients the Neck Linker of Kinesin in Two Sequential Steps* , 2001, The Journal of Biological Chemistry.

[16]  Ronald D Vale,et al.  The Molecular Motor Toolbox for Intracellular Transport , 2003, Cell.

[17]  A. Houdusse,et al.  Atomic Structure of Scallop Myosin Subfragment S1 Complexed with MgADP A Novel Conformation of the Myosin Head , 1999, Cell.

[18]  H. Higuchi,et al.  Rapid double 8‐nm steps by a kinesin mutant , 2004, The EMBO journal.

[19]  S. Ishiwata,et al.  Nucleotide-dependent single- to double-headed binding of kinesin. , 2001, Science.

[20]  T. Yanagida,et al.  Fluorescence resonance energy transfer between single fluorophores attached to a coiled-coil protein in aqueous solution , 1999 .

[21]  R. Vale,et al.  The way things move: looking under the hood of molecular motor proteins. , 2000, Science.

[22]  Ronald D. Vale,et al.  Role of the kinesin neck linker and catalytic core in microtubule-based motility , 2000, Current Biology.

[23]  Roger Cooke,et al.  Two conformations in the human kinesin power stroke defined by X-ray crystallography and EPR spectroscopy , 2002, Nature Structural Biology.

[24]  E. Mandelkow,et al.  X-ray structure of motor and neck domains from rat brain kinesin. , 1997, Biochemistry.

[25]  Steven M. Block,et al.  Kinesin Moves by an Asymmetric Hand-OverHand Mechanism , 2003 .

[26]  Masahide Kikkawa,et al.  Switch-based mechanism of kinesin motors , 2001, Nature.

[27]  Ryo Nitta,et al.  KIF1A Alternately Uses Two Loops to Bind Microtubules , 2004, Science.

[28]  Hernando Sosa,et al.  Nucleotide binding and hydrolysis induces a disorder-order transition in the kinesin neck-linker region , 2006, Nature Structural &Molecular Biology.

[29]  M. Sheetz,et al.  Tracking kinesin-driven movements with nanometre-scale precision , 1988, Nature.

[30]  J Guo,et al.  Crystal Structure of the Mitotic Spindle Kinesin Eg5 Reveals a Novel Conformation of the Neck-linker* , 2001, The Journal of Biological Chemistry.

[31]  D. Hackney,et al.  The tethered motor domain of a kinesin-microtubule complex catalyzes reversible synthesis of bound ATP. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[32]  K. Sutoh,et al.  Swing of the lever arm of a myosin motor at the isomerization and phosphate-release steps , 1998, Nature.

[33]  Hernando Sosa,et al.  Configuration of the two kinesin motor domains during ATP hydrolysis , 2003, Nature Structural Biology.

[34]  Christoph F. Schmidt,et al.  Direct observation of kinesin stepping by optical trapping interferometry , 1993, Nature.

[35]  Toshio Yanagida,et al.  Direct observation of single kinesin molecules moving along microtubules , 1996, Nature.

[36]  Hiroyasu Itoh,et al.  How two-foot molecular motors may walk. , 2005, Advances in experimental medicine and biology.

[37]  Ronald D. Vale,et al.  Crystal structure of the kinesin motor domain reveals a structural similarity to myosin , 1996, Nature.

[38]  Ronald D Vale,et al.  Conversion of Unc104/KIF1A Kinesin into a Processive Motor After Dimerization , 2002, Science.

[39]  S. Rosenfeld,et al.  Measuring Kinesin's First Step* , 2002, The Journal of Biological Chemistry.

[40]  A. Skerra,et al.  Applications of a peptide ligand for streptavidin: the Strep-tag. , 1999, Biomolecular engineering.

[41]  Hideo Higuchi,et al.  Alternate fast and slow stepping of a heterodimeric kinesin molecule , 2003, Nature Cell Biology.

[42]  J. Gelles,et al.  Distinguishing Inchworm and Hand-Over-Hand Processive Kinesin Movement by Neck Rotation Measurements , 2002, Science.