View the Current Issue

Article

  • The EMBO Journal (2003) 22, 1518 - 1528
  • doi:10.1093/emboj/cdg164

Nucleotide-induced conformations in the neck region of dimeric kinesin

Georgios Skiniotis1, Thomas Surrey1, Stephan Altmann1, Heinz Gross2, Young-Hwa Song3, Eckhard Mandelkow3 and Andreas Hoenger1

  1. European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
  2. Institute of Cell Biology, Swiss Federal Institute of Technology, CH-8093 Zürich-Hoenggerberg, Switzerland
  3. Max Planck Unit for Structural Molecular Biology, c/o DESY-Hamburg, Notkestrasse 85, D-22607, Hamburg, Germany

Correspondence to:

Andreas Hoenger, E-mail: hoenger@embl-heidelberg.de

Received 25 October 2002; Accepted 14 February 2003; Revised 10 February 2003

The neck region of kinesin constitutes a key component in the enzyme's walking mechanism. Here we applied cryoelectron microscopy and image reconstruction to investigate the location of the kinesin neck in dimeric and monomeric constructs complexed to microtubules. To this end we enhanced the visibility of this region by engineering an SH3 domain into the transition between neck linker and neck coiled coil. The resulting chimeric kinesin constructs remained functional as verified by physiology assays. In the presence of AMP–PNP the SH3 domains allowed us to identify the position of the neck in a well defined conformation and revealed its high flexibility in the absence of nucleotide. We show here the double-headed binding of dimeric kinesin along the same protofilament, which is characterized by the opposite directionality of neck linkers. In this configuration the neck coiled coil appears fully zipped. The position of the neck region in dimeric constructs is not affected by the presence of the tubulin C-termini as confirmed by subtilisin treatment of microtubules prior to motor decoration.

  • Keywords:

    • cryoelectron microscopy,
    • kinesin,
    • kinesin neck,
    • microtubules,
    • SH3 domain