A new look at the microtubule binding patterns of dimeric kinesins

The interactions of monomeric and dimeric kinesin and ncd constructs with m icrotubules have been investigated using cryo-electron microscopy (cryo-EM) and several biochemical methods. There is a good consensus on the structur e of dimeric ncd when bound to a tubulin dimer showing one head attached di rectly to tubulin, and the second head tethered to the first. However, the 3D maps of dimeric kinesin motor domains are still quite controversial and leave room for different interpretations. Here we reinvestigated the microt ubule binding patterns of dimeric kinesins by cryo-EM and digital 3D recons truction under different nucleotide conditions and different motor:tubulin ratios, and determined the molecular mass of motor-tubulin complexes by STE M. Both methods revealed complementary results. We found that the ratio of bound kinesin motor-heads to ap-tubulin dimers was never reaching above 1.5 irrespective of the initial mixing ratios. It appears that each kinesin di mer occupies two microtubule-binding sites, provided that there is a free o ne nearby. Thus the appearances of different image reconstructions can be e xplained by non-specific excess binding of motor heads. Consequently, the u se of different apparent density distributions for docking the X-ray struct ures onto the microtubule surface leads to different and mutually exclusive models. We propose that in conditions of stoichiometric binding the two he ads of a kinesin dimer separate and bind to different tubulin subunits. Thi s is in contrast to ncd where the two heads remain tightly attached on the microtubule surface. Using dimeric kinesin molecules crosslinked in their n eck domain we also found that they stabilize protofilaments axially, but no t laterally, which is a strong indication that the two heads of the dimers bind along one protofilament, rather than laterally bridging two protofilam ents. A molecular walking model based on these results summarizes our concl usions and illustrates the implications of symmetry for such models. (C) 20 00 Academic Press.