Intramolecular Strain Coordinates Kinesin Stepping Behavior along Microtubules

Kinesin advances 8 nm along a microtubule per ATP hydrolyzed, but the mechanism responsible for coordinating the enzymatic cycles of kinesin's two identical motor domains remains unresolved. Here, we have tested whether such coordination is mediated by intramolecular tension generated by the "neck linkers," mechanical elements that span between the motor domains. When tension is reduced by extending the neck linkers with artificial peptides, the coupling between ATP hydrolysis and forward stepping is impaired and motor's velocity decreases as a consequence. However, speed recovers to nearly normal levels when external tension is applied by an optical trap. Remarkably, external load also induces bidirectional stepping of an immotile kinesin that lacks its mechanical element (neck linker) and fuel (ATP). Our results indicate that the kinesin motor domain senses and responds to strain in a manner that facilitates its plus-end-directed stepping and communication between its two motor domains.

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

[2]  R. Cross,et al.  What kinesin does at roadblocks: the coordination mechanism for molecular walking , 2004, The EMBO journal.

[3]  W. Eaton,et al.  Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Steven M Block,et al.  Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Spudich.  Molecular Motors Take Tension in Stride , 2006, Cell.

[6]  Matthias Rief,et al.  Myosin-V is a mechanical ratchet. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Samara L. Reck-Peterson,et al.  Force-Induced Bidirectional Stepping of Cytoplasmic Dynein , 2007, Cell.

[8]  Julia Hoeng,et al.  An ATP Gate Controls Tubulin Binding by the Tethered Head of Kinesin-1 , 2007, Science.

[9]  Ram Dixit,et al.  Differential Regulation of Dynein and Kinesin Motor Proteins by Tau , 2008, Science.

[10]  P. Schimmel,et al.  Conformational energy and configurational statistics of poly-L-proline. , 1967, Proceedings of the National Academy of Sciences of the United States of America.

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

[12]  Steven M. Block,et al.  Force and velocity measured for single kinesin molecules , 1994, Cell.

[13]  J. Howard,et al.  Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

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

[15]  Steven M Block,et al.  Kinesin motor mechanics: binding, stepping, tracking, gating, and limping. , 2007, Biophysical journal.

[16]  Nico Stuurman,et al.  Single-molecule observations of neck linker conformational changes in the kinesin motor protein , 2006, Nature Structural &Molecular Biology.

[17]  M. Schnitzer,et al.  Statistical kinetics of processive enzymes. , 1995, Cold Spring Harbor symposia on quantitative biology.

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

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

[20]  R. Vale,et al.  Controlling Kinesin by Reversible Disulfide Cross-linking : Identifying the Motility-producing Conformational Change ✪ , 2000 .

[21]  Shin'ichi Ishiwata,et al.  Loading direction regulates the affinity of ADP for kinesin , 2003, Nature Structural Biology.

[22]  I. Gibbons,et al.  A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella. , 1979, The Journal of biological chemistry.

[23]  P. Cowan,et al.  The Polypeptide Chain Configuration of Collagen , 1955, Nature.

[24]  Changbong Hyeon,et al.  Internal strain regulates the nucleotide binding site of the kinesin leading head , 2007, Proceedings of the National Academy of Sciences.

[25]  Amber L. Wells,et al.  Myosin VI is a processive motor with a large step size , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Dietmar Labeit,et al.  Molecular Mechanics of Cardiac Titin's PEVK and N2B Spring Elements* , 2002, The Journal of Biological Chemistry.

[27]  Martin Zacharias,et al.  Distance distributions of short polypeptides recovered by fluorescence resonance energy transfer in the 10 A domain. , 2006, Journal of the American Chemical Society.

[28]  Joshua W. Shaevitz,et al.  Probing the kinesin reaction cycle with a 2D optical force clamp , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Megan T Valentine,et al.  To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5. , 2007, Current opinion in cell biology.

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

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

[32]  Alex Mogilner,et al.  Mechanics of Motor Proteins and the Cytoskeleton , 2002 .

[33]  Martin Karplus,et al.  Force generation in kinesin hinges on cover-neck bundle formation. , 2008, Structure.

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

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

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

[37]  Clara Franzini-Armstrong,et al.  A flexible domain is essential for the large step size and processivity of myosin VI. , 2005, Molecular cell.

[38]  Liedewij Laan,et al.  Assembly dynamics of microtubules at molecular resolution , 2006, Nature.

[39]  R. Vale,et al.  How kinesin waits between steps , 2007, Nature.

[40]  K. Johnson,et al.  Alternating site mechanism of the kinesin ATPase. , 1998, Biochemistry.

[41]  E. Siggia,et al.  Entropic elasticity of lambda-phage DNA. , 1994, Science.

[42]  K. Svoboda,et al.  Fluctuation analysis of motor protein movement and single enzyme kinetics. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[43]  Paul R. Selvin,et al.  Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization , 2003, Science.

[44]  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.

[45]  R A Milligan,et al.  Kinesin follows the microtubule's protofilament axis , 1993, The Journal of cell biology.

[46]  Ronald D. Vale,et al.  Engineering the Processive Run Length of the Kinesin Motor , 2000, The Journal of cell biology.

[47]  Marko,et al.  Statistical mechanics of supercoiled DNA. , 1995, Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics.

[48]  D. Hackney.  The rate-limiting step in microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule. , 1994, The Journal of biological chemistry.

[49]  J. Howard,et al.  Kinesin's processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

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

[51]  Andreas Hoenger,et al.  Kinesin's second step. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[54]  Toshio Yanagida,et al.  Chemomechanical coupling of the forward and backward steps of single kinesin molecules , 2002, Nature Cell Biology.

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

[56]  M. F. Stock,et al.  Modulation of kinesin half-site ADP release and kinetic processivity by a spacer between the head groups. , 2003, Biochemistry.

[57]  Mark J. Schnitzer,et al.  Kinesin hydrolyses one ATP per 8-nm step , 1997, Nature.

[58]  Samara L. Reck-Peterson,et al.  Single-Molecule Analysis of Dynein Processivity and Stepping Behavior , 2006, Cell.