Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain.

The two-headed kinesin motor harnesses the energy of ATP hydrolysis to take 8-nm steps, walking processively along a microtubule, alternately stepping with each of its catalytic heads in a hand-over-hand fashion. Two persistent challenges for models of kinesin motility are to explain how the two heads are coordinated ("gated") and when the translocation step occurs relative to other events in the mechanochemical reaction cycle. To investigate these questions, we used a precision optical trap to measure the single-molecule kinetics of kinesin in the presence of substrate analogs beryllium fluoride or adenylyl-imidodiphosphate. We found that normal stepping patterns were interspersed with long pauses induced by analog binding, and that these pauses were interrupted by short-lived backsteps. After a pause, processive stepping could only resume once the kinesin molecule took an obligatory, terminal backstep, exchanging the positions of its front and rear heads, presumably to allow release of the bound analog from the new front head. Preferential release from the front head implies that the kinetics of the two heads are differentially affected when both are bound to the microtubule, presumably by internal strain that is responsible for the gating. Furthermore, we found that ATP binding was required to reinitiate processive stepping after the terminal backstep. Together, our results support stepping models in which ATP binding triggers the mechanical step and the front head is gated by strain.

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

[2]  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.

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

[4]  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.

[5]  J. Gelles,et al.  Coupling of kinesin steps to ATP hydrolysis , 1997, Nature.

[6]  Michael P. Sheetz,et al.  Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility , 1985, Cell.

[7]  R. Vale,et al.  Chemomechanical cycle of kinesin differs from that of myosin , 1993, Nature.

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

[9]  A. Hudspeth,et al.  Movement of microtubules by single kinesin molecules , 1989, Nature.

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

[11]  K. Johnson,et al.  Pre-steady-state kinetics of the microtubule-kinesin ATPase. , 1994, Biochemistry.

[12]  M. Sheetz,et al.  Delayed start-up of kinesin-driven microtubule gliding following inhibition by adenosine 5'-[beta,gamma-imido]triphosphate. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[13]  J. Spudich,et al.  From the Cover : A force-dependent state controls the coordination of processive myosin V , 2005 .

[14]  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.

[15]  E. Taylor,et al.  Mechanism of microtubule kinesin ATPase. , 1995, Biochemistry.

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

[17]  S. Rosenfeld,et al.  Equilibrium Studies of Kinesin-Nucleotide Intermediates (*) , 1996, The Journal of Biological Chemistry.

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

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

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

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

[22]  I. Rayment,et al.  X-ray structures of the MgADP, MgATPgammaS, and MgAMPPNP complexes of the Dictyostelium discoideum myosin motor domain. , 1997, Biochemistry.

[23]  S. Ishiwata,et al.  Thermal activation of single kinesin molecules with temperature pulse microscopy. , 2001, Cell motility and the cytoskeleton.

[24]  P. Kollman,et al.  Closing of the Nucleotide Pocket of Kinesin-Family Motors upon Binding to Microtubules , 2003, Science.

[25]  L. Goldstein,et al.  Bead movement by single kinesin molecules studied with optical tweezers , 1990, Nature.

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

[27]  H M Holden,et al.  X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP.BeFx and MgADP.AlF4-. , 1995, Biochemistry.

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

[29]  E. Taylor,et al.  Interacting Head Mechanism of Microtubule-Kinesin ATPase* , 1997, The Journal of Biological Chemistry.

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

[31]  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.

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

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

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

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

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

[37]  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.

[38]  E. Taylor,et al.  Kinetic Mechanism of a Monomeric Kinesin Construct* , 1997, The Journal of Biological Chemistry.

[39]  Shin'ichi Ishiwata,et al.  Kinesin–microtubule binding depends on both nucleotide state and loading direction , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[40]  Anne Houdusse,et al.  Three myosin V structures delineate essential features of chemo‐mechanical transduction , 2004, The EMBO journal.

[41]  S. Block,et al.  Versatile optical traps with feedback control. , 1998, Methods in enzymology.

[42]  W E Moerner,et al.  Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules. , 2001, Biophysical journal.

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

[44]  S. Block,et al.  Kinesin: What Gives? , 1998, Cell.

[45]  S. Rosenfeld,et al.  Magnesium Regulates ADP Dissociation from Myosin V* , 2005, Journal of Biological Chemistry.

[46]  R. Lasek,et al.  Attachment of transported vesicles to microtubules in axoplasm is facilitated by AMP-PNP , 1985, Nature.

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

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

[49]  J. Sellers,et al.  Load-dependent kinetics of myosin-V can explain its high processivity , 2005, Nature Cell Biology.

[50]  E. Mandelkow,et al.  The structural and mechanochemical cycle of kinesin. , 1998, Trends in biochemical sciences.

[51]  Susan P. Gilbert,et al.  Pathway of processive ATP hydrolysis by kinesin , 1995, Nature.

[52]  S. Auerbach,et al.  Alternating Site ATPase Pathway of Rat Conventional Kinesin* , 2005, Journal of Biological Chemistry.

[53]  S. Maruta,et al.  Formation and characterization of kinesin.ADP.fluorometal complexes. , 2002, Journal of biochemistry.