Kinesin's cover-neck bundle folds forward to generate force

Each step of the kinesin motor involves a force-generating molecular rearrangement. Although significant progress has been made in elucidating the broad features of the kinesin mechanochemical cycle, molecular details of the force generation mechanism remain a mystery. Recent molecular dynamics simulations have suggested a mechanism in which the forward drive is produced when the N-terminal cover strand forms a β-sheet with the neck linker to yield the cover-neck bundle. We tested this proposal by comparing optical trapping motility measurements of cover strand mutants with the wild-type. Motility data, as well as kinetic analyses, revealed impairment of the force-generating capacity accompanied by a greater load dependence in the mechanochemical cycle. In particular, a mutant with the cover strand deleted functioned only marginally, despite the fact that the cover strand, the N-terminal “dangling end,” unlike the neck linker and nucleotide-binding pocket, is not involved with any previously considered energy transduction pathway. Furthermore, a constant assisting load, likely in lieu of a power stroke, was shown to rescue forward motility in the cover strand deletion mutant. Our results support a stepping mechanism driven by dynamic cover-neck bundle formation. They also suggest a strategy to generate motors with altered mechanical characteristics by targeting the force-generating element.

[1]  S. Englander,et al.  The N-terminal to C-terminal motif in protein folding and function. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Gelles,et al.  Failure of a single-headed kinesin to track parallel to microtubule protofilaments , 1995, Nature.

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

[4]  S. Endow,et al.  Determinants of kinesin motor polarity. , 1998, Science.

[5]  A. Kolomeisky,et al.  Simple mechanochemistry describes the dynamics of kinesin molecules , 2001, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[8]  Joshua W Shaevitz,et al.  An automated two-dimensional optical force clamp for single molecule studies. , 2002, Biophysical journal.

[9]  Ahmad S. Khalil,et al.  Single M13 bacteriophage tethering and stretching , 2007, Proceedings of the National Academy of Sciences.

[10]  James A. Spudich,et al.  The Mechanism of Myosin VI Translocation and Its Load-Induced Anchoring , 2004, Cell.

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

[12]  Michelle D. Wang,et al.  Force and velocity measured for single molecules of RNA polymerase. , 1998, Science.

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

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

[15]  Crystal structure of the kinesin motor domain reveals a structural similarity to myosin , 1996 .

[16]  Polly M. Fordyce,et al.  Stepping and Stretching , 2003, The Journal of Biological Chemistry.

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

[18]  Masahide Kikkawa,et al.  The role of microtubules in processive kinesin movement. , 2008, Trends in cell biology.

[19]  K. Svoboda,et al.  Biological applications of optical forces. , 1994, Annual review of biophysics and biomolecular structure.

[20]  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.

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

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

[23]  J. Cronan,et al.  Microtubule movement by a biotinated kinesin bound to streptavidin-coated surface. , 1994, The Journal of biological chemistry.

[24]  Kenneth C Holmes,et al.  The molecular mechanism of muscle contraction. , 2005, Advances in protein chemistry.

[25]  Kenneth H. Downing,et al.  The beginning of kinesin's force-generating cycle visualized at 9-Å resolution , 2007, The Journal of cell biology.

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

[27]  A B Kolomeisky,et al.  The force exerted by a molecular motor. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[30]  T. Yanagida,et al.  Mechanics of single kinesin molecules measured by optical trapping nanometry. , 1997, Biophysical journal.

[31]  Ulrich H E Hansmann,et al.  Simulation of Top7-CFr: A transient helix extension guides folding , 2008, Proceedings of the National Academy of Sciences.

[32]  W. Hwang.  Calculation of conformation-dependent biomolecular forces. , 2007, The Journal of chemical physics.