SUBUNIT INTERACTIONS IN DIMERIC KINESIN HEAVY-CHAIN DERIVATIVES THAT LACK THE KINESIN ROD

The N-terminal residues of the two heavy chains of the motor enzyme kinesin form two globular ''heads''; the heads are attached to a ''rod'' domain which is a two-stranded cu-helical colied-coli, Interaction between the heads is thought to be important to kinesin function, The rod may not be necessary for head-head interactions because a heavy chain N-terminal fragment containing only residues from the head and adjacent region forms dimers (Huang, T,-G,, Suhan, J,, and Hackney, D, D, (1994) J, Biol. Chem. 269, 16502-16507), However, the nature and stability of the subunit-subunit interactions in such derivatives are unclear, To examine the physical properties of heavy chain interaction in and near the head domains, we characterized the self-association behavior of two dimeric kinesin derivatives predicted (Lupas, k, van Dyke, Ri., and Stock, J, (1991) Science 252, 1162-1164) to lack the rod, Derivative K448-BIO contains the 448 N-terminal residues of Drosophila kinesin heavy chain fused at the C terminus to a a-residue Linker and a C-terminal fragment from Escherichia coli biotin carboxyl carrier protein; derivative K448-L is the same except that it lacks the biotin carboxyl carrier protein fragment, Both derivatives expressed in insect cells display microtubule-stimulated ATPase activity; K448-BIO also displays microtubule motility, Equilibrium sedimentation and gel filtration indicate that purified K448-BIO and H448-L at 0.02-0.4 mg/ml form homogeneous solutions of homodimers with no detectable formation of monomers or higher order oligomers, Derivative self-association is non covalent but extremely stable with an association constant greater than or equal to 2 x 10(8) M(-1). Stable subunit-subunit association induced by structures in and near the kinesin heads may be necessary for full mechanochemical function.