All Title Author
Keywords Abstract

PLOS ONE  2012 

Altered Nucleotide-Microtubule Coupling and Increased Mechanical Output by a Kinesin Mutant

DOI: 10.1371/journal.pone.0047148

Full-Text   Cite this paper   Add to My Lib

Abstract:

Kinesin motors hydrolyze ATP to produce force and do work in the cell – how the motors do this is not fully understood, but is thought to depend on the coupling of ATP hydrolysis to microtubule binding by the motor. Transmittal of conformational changes from the microtubule- to the nucleotide-binding site has been proposed to involve the central β-sheet, which could undergo large structural changes important for force production. We show here that mutation of an invariant residue in loop L7 of the central β-sheet of the Drosophila kinesin-14 Ncd motor alters both nucleotide and microtubule binding, although the mutated residue is not present in either site. Mutants show weak-ADP/tight-microtubule binding, instead of tight-ADP/weak-microtubule binding like wild type – they hydrolyze ATP faster than wild type, move faster in motility assays, and assemble long spindles with greatly elongated poles, which are also produced by simulations of assembly with tighter microtubule binding and faster sliding. The mutated residue acts like a mechanochemical coupling element – it transmits changes between the microtubule-binding and active sites, and can switch the state of the motor, increasing mechanical output by the motor. One possibility, based on our findings, is that movements by the residue and the loop that contains it could bend or distort the central β-sheet, mediating free energy changes that lead to force production.

References

[1]  Hirose K, Akimaru E, Akiba T, Endow SA, Amos LA (2006) Large conformational changes in a kinesin motor catalysed by interaction with microtubules. Mol Cell 23: 913–923.
[2]  Howard J (2001) Mechanics of motor proteins and the cytoskeleton. Sunderland, MA: Sinauer Associates, Inc. 367 p.
[3]  Kikkawa M, Sablin EP, Okada Y, Yajima H, Fletterick RJ, et al. (2001) Switch-based mechanism of kinesin motors. Nature 411: 439–445.
[4]  Kikkawa M, Hirokawa N (2006) High-resolution cryo-EM maps show the nucleotide binding pocket of KIF1A in open and closed conformations. EMBO J 25: 4187–4194.
[5]  Sindelar CV, Downing KH (2010) An atomic-level mechanism for activation of the knesin molecular motors. Proc Natl Acad Sci USA 107: 4111–4116.
[6]  Kozielski F, De Bonis S, Burmeister WP, Cohen-Addad C, Wade RH (1999) The crystal structure of the minus-end-directed microtubule motor protein ncd reveals variable dimer conformations. Structure 7: 1407–1416.
[7]  Liu H-L, Pemble IV CW, Endow SA (2012) Neck-motor interactions trigger rotation of the kinesin stalk. Sci Rep 2: 236.
[8]  Nitta R, Okada Y, Hirokawa N (2008) Structural model for strain-dependent microtubule activation of Mg-ADP release from kinesin. Nat Struct Mol Biol 15: 1067–1075.
[9]  Pechatnikova E, Taylor EW (1999) Kinetics processivity and the direction of motion of Ncd. Biophys J 77: 1003–1016.
[10]  Hirose K, Cross RA, Amos LA (1998) Nucleotide-dependent structural changes in dimeric NCD molecules complexed to microtubules. J Mol Biol 278: 389–400.
[11]  Crevel IM-TC, Lockhart A, Cross RA (1996) Weak and strong states of kinesin and ncd. J Mol Biol 257: 66–76.
[12]  Heuston E, Bronner CE, Kull FJ, Endow SA (2010) A kinesin motor in a force-producing conformation. BMC Struct Biol 10: 19.
[13]  Yamamoto AH, Komma DJ, Shaffer CD, Pirrotta V, Endow SA (1989) The claret locus in Drosophila encodes products required for eyecolor and for meiotic chromosome segregation. EMBO J 8: 3543–3552.
[14]  Sk?ld HN, Komma DJ, Endow SA (2005) Assembly pathway of the anastral Drosophila oocyte meiosis I spindle. J Cell Sci 118: 1745–1755.
[15]  Matthies HJG, McDonald HB, Goldstein LSB, Theurkauf WE (1996) Anastral meiotic spindle morphogenesis: role of the Non-Claret Disjunctional kinesin-like protein. J Cell Biol 134: 455–464.
[16]  Endow SA, Komma DJ (1997) Spindle dynamics during meiosis in Drosophila oocytes. J Cell Biol 137: 1321–1336.
[17]  Surrey T, Nedelec F, Leibler S, Karsenti E (2001) Physical properties determining self-organization of motors and microtubules. Science 292: 1167–1171.
[18]  Hallen MA, Endow SA (2009) Anastral spindle assembly: a mathematical model. Biophys J 97: 2191–2201.
[19]  Burbank KS, Groen AC, Perlman ZE, Fisher DS, Mitchison TJ (2006) A new method reveals microtubule minus ends throughout the meiotic spindle. J Cell Biol 175: 369–375.
[20]  Dumont S, Mitchison TJ (2009) Force and length in the mitotic spindle. Curr Biol 19: R749–R761.
[21]  Liang Z-Y, Hallen MA, Endow SA (2009) Mature Drosophila meiosis I spindles comprise microtubules of mixed polarity. Curr Biol 19: 163–168.
[22]  Yang G, Houghtaling BR, Gaetz J, Liu JZ, Danuser G, et al. (2007) Architectural dynamics of the meiotic spindle revealed by single-fluorophore imaging. Nature Cell Biol 9: 1233–1242.
[23]  Song H, Endow SA (1998) Decoupling of nucleotide- and microtubule-binding in a kinesin mutant. Nature 396: 587–590.
[24]  Higuchi H, Bronner CE, Park HW, Endow SA (2004) Rapid double 8-nm steps by a kinesin mutant. EMBO J 23: 2993–2999.
[25]  Yun M, Bronner CE, Park C-G, Cha S-S, Park H-W, et al. (2003) Rotation of the stalk/neck and one head in a new crystal structure of the kinesin motor protein, Ncd. EMBO J 22: 5382–5389.
[26]  Grant BJ, Gheorghe DM, Zheng W, Alonso M, Huber G, et al. (2011) Electrostatically biased binding of kinesin to microtubules. PLos Biology 9: e1001207.
[27]  Muller HJ (1932) Further studies on the nature and causes of gene mutations. Proc 6th Internat Congress Genet 1: 213–255.
[28]  Herskowitz I (1987) Functional inactivation of genes by dominant negative mutations. Nature 329: 219–222.
[29]  Reubold TF, Eschenburg S, Becker A, Kull FJ, Manstein DJ (2003) A structural model for actin-induced nucleotide release in myosin. Nature Struct Biol 10: 826–830.
[30]  Coureux P-D, Sweeney HL, Houdusse A (2004) Three myosin V structures delineate essential features of chemo-mechanical transduction. EMBO J 23: 4527–4537.
[31]  Way M, Pope P, Gooch J, Hawkins M, Weeds AG (1990) Identification of a region in segment 1 of gelsolin critical for actin binding. EMBO J 9: 4103–4109.
[32]  Song H, Endow SA (1997) Rapid purification of microtubule motor domain proteins expressed in bacteria. Bio Techniques 22: 82–85.
[33]  Huang T-G, Hackney DD (1994) Drosophila kinesin minimal motor domain expressed in Escherichia coli. J Biol Chem 269: 16493–16501.
[34]  Wagenbach M, Domnitz S, Wordeman L, Cooper J (2008) A kinesin-13 mutant catalytically depolymerizes microtubules in ADP. J Cell Biol 183: 617–623.
[35]  Rasband W (1997–2008) ImageJ. 1.45s ed. National Institutes of Health, USA: http://imagej.nih.gov/ij.
[36]  Song H, Golovkin M, Reddy ASN, Endow SA (1997) In vitro motility of AtKCBP, a calmodulin-binding kinesin protein of Arabidopsis. Proc Natl Acad Sci USA 94: 322–327.
[37]  Endow SA, Komma DJ (1996) Centrosome and spindle function of the Drosophila Ncd microtubule motor visualized in live embryos using Ncd-GFP fusion proteins. J Cell Sci 109: 2429–2442.
[38]  Zou J, Hallen MA, Yankel CD, Endow SA (2008) A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes. J Cell Biol 180: 459–466.
[39]  DeLano WL (2002) The PyMOL Molecular Graphics System. San Carlos, CA: DeLano Scientific.
[40]  Hallen MA, Liang Z-Y, Endow SA (2011) Two-state displacement by the kinesin-14 Ncd stalk. Biophys Chem 154: 56–65.
[41]  Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, et al. (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 25: 1605–1612.

Full-Text

comments powered by Disqus