Evidence that the stalk of Drosophila kinesin heavy chain is an alpha-helical coiled coil.
Kinesin is a mechanochemical enzyme composed of three distinct domains: a globular head domain, a rodlike stalk domain, and a small globular tail domain. The stalk domain has sequence features characteristic of alpha-helical coiled coils. To gain insight into the structure of the kinesin stalk, we expressed it from a segment of the Drosophila melanogaster kinesin heavy chain gene and purified it from Escherichia coli. When observed by EM, this protein formed a rodlike structure 40-55 nm long that was occasionally bent at a hingelike region near the middle of the molecule. An additional EM study and a chemical cross-linking study showed that this protein forms a parallel dimer and that the two chains are in register. Finally, using circular dichroism spectroscopy, we showed that this protein is approximately 55-60% alpha-helical in physiological aqueous solution at 25 degrees C, and approximately 85-90% alpha-helical at 4 degrees C. From these results, we conclude that the stalk of kinesin heavy chain forms an alpha-helical coiled coil structure. The temperature dependence of the circular dichroism signal has two major transitions, at 25-30 degrees C and at 45-50 degrees C, which suggests that a portion of the alpha-helical structure in the stalk is less stable than the rest. By producing the amino-terminal (coil 1) and carboxy-terminal (coil 2) halves of the stalk separately in E. coli, we showed that the region that melts below 30 degrees C lies within coil 1, while the majority of coil 2 melts above 45 degrees C. We suggest that this difference in stability may play a role in the force-generating mechanism or regulation of kinesin.
de Cuevas
M
M
Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138.
Tao
T
T
Goldstein
L S
LS
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D004228:Dithionitrobenzoic Acid; D004331:Drosophila melanogaster / Q000737:chemistry*; D016547:Kinesins / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D013696:Temperature
D011993:Recombinant Fusion Proteins; D004228:Dithionitrobenzoic Acid; D016547:Kinesins