Three-dimensional structure of functional motor proteins on microtubules.
BACKGROUND
Kinesins are a superfamily of motor proteins that use ATP hydrolysis to fuel movement along microtubules and participate in many crucial phases of the eukaryotic cell cycle. Usually these motors are heterotetramers of two heavy and two light chains, and have globular motor domains on the two heavy chains. Most kinesins move towards the microtubule 'plus end', but some, such as ncd (nonclaret disjunctional protein), move in the opposite direction. Heavy chain dimers produced by overexpression are viable motors.


RESULTS
In order to establish whether the opposite directionality of kinesin and ncd dimers is related to notable conformational differences, we have used electron cryo-microscopy and three-dimensional reconstruction methods to investigate the structure of kinesin and ncd dimers attached to microtubules in the presence of AMP-PNP (5'-adenylylimidodiphosphate), a nonhydrolyzable ATP analogue. Three-dimensional maps of the motor-microtubule complexes show the motors to have one unattached, and one attached head per tubulin dimer. The polarity of the reconstructions was determined for each individual microtubule. Attachment occurs on the crest of a protofilament at the end of the tubulin dimer that points towards the plus end of the microtubule. The attached head extends over the next tubulin molecule along the protofilament. The unattached heads of kinesin and ncd have distinctly different conformations.


CONCLUSIONS
The attached heads of kinesin and ncd appear to be similar and to interact with the same region of the plus end-oriented tubulin subunits. The free heads, however, are quite different, which suggests that directionality could be determined by differences in the dimer conformations. Work is in progress to obtain three-dimensional maps in the presence of different nucleotides with the aim of understanding how these motors move along microtubules.
Arnal
I
I
Laboratoire de Microscopie Electronique Structurale, Institut de Biologie Structurale, Grenoble, France.
Metoz
F
F
DeBonis
S
S
Wade
R H
RH
D000818:Animals; D002417:Cattle; D004330:Drosophila; D029721:Drosophila Proteins; D007091:Image Processing, Computer-Assisted; D016547:Kinesins / Q000737:chemistry*; D008868:Microtubule Proteins / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry* / Q000648:ultrastructure; D011487:Protein Conformation; D014404:Tubulin / Q000737:chemistry
D029721:Drosophila Proteins; D008868:Microtubule Proteins; D014404:Tubulin; C066696:ncd protein, Drosophila; D016547:Kinesins