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 approximately 120 kDa and two light chains of approximately 65 kDa protein. Kinesin motor activity is dependent on the presence of ATP and microtubules. The kinesin light chain-binding site in human kinesin heavy chain was determined by reconstituting in vitro a complex of recombinant heavy and light chains. The proteins expressed in bacteria included oligohistidine-tagged fragments of human ubiquitous kinesin heavy chain, spanning most of the stalk and all of the tail domain (amino acids 555-963); and untagged, essentially full-length human kinesin light chain (4-569) along with N-terminal (4-363) and C-terminal (364-569) light chain fragments. Heavy chain fragments were attached to Ni2+-charged beads and incubated with untagged light chain fragments. Analysis of eluted complexes by SDS-PAGE and immunoblotting mapped the light chain-binding site in heavy chain to amino acids 771-813, a region close to the C-terminal end of the heavy chain stalk domain. In addition, 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 alpha-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 kinesin heavy chain fragment 771-963, which displays an alpha-helical content of 70%, and the resistance of the heavy/light chain interaction to high salt (0.5 M).
Diefenbach
R J
RJ
Centre for Virus Research, Westmead Institutes of Health Research, Westmead Hospital, NSW, Australia. russelld@westgate.wh.usyd.edu.au
Mackay
J P
JP
Armati
P J
PJ
Cunningham
A L
AL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D049832:Decapodiformes; D004330:Drosophila; D006801:Humans; D016547:Kinesins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012617:Sea Urchins
D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016547:Kinesins