The C-terminal region of the stalk domain of ubiquitous human kinesin heavy chain contains the binding site for kinesin light chain

The motor protein kinesin is a heterotetramer composed of two heavy chains of similar to 120 kDa and two light chains of similar to 65 kDa protein. Ki nesin motor activity is dependent on the presence of ATP and microtubules. The kinesin light chain-binding site in human kinesin heavy chain was deter mined by reconstituting in vitro a complex of recombinant heavy and light c hains. The proteins expressed in bacteria included oligohistidine-tagged fr agments of human ubiquitous kinesin heavy chain, spanning most of the stalk and all of the tail domain (amino acids 555-963); and untagged, essentiall y full-length human kinesin light chain (4-569) along with N-terminal (4-36 3) and C-terminal (364-569) light chain fragments. Heavy chain fragments we re attached to Ni2+-charged beads and incubated with untagged light chain f ragments. Analysis of eluted complexes by SDS-PAGE and immunoblotting mappe d the light chain-binding site in heavy chain to amino acids 771-813, a reg ion close to the C-terminal end of the heavy chain stalk domain. In additio n, only the full-length and N-terminal kinesin light chain fragments bound to this heavy chain region. Within this heavy chain region are four highly conserved contiguous heptad repeats (775-802) which are predicted to form a tight a-helical coiled-coil interaction with the heptad repeat-containing N-terminus of the light chain, in particular region 106-152 of human Light chain. This predicted hydrophobic, alpha-helical coiled-coil interaction is supported by both circular dichroism spectroscopy of the recombinant kines in heavy chain fragment 771-963, which displays an a-helical content of 70% , and the resistance of the heavy/light chain interaction to high salt (0.5 M).