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 alpha-helical coiled-coil. 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, A., van Dyke, M., 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 2-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 K448-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 > or = 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.
Young
E C
EC
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Berliner
E
E
Mahtani
H K
HK
Perez-Ramirez
B
B
Gelles
J
J
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D004220:Disulfides; D004330:Drosophila; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011487:Protein Conformation; D018411:Spodoptera
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D004220:Disulfides; D016547:Kinesins