Evidence that the stalk of Drosophila kinesin heavy chain is an α-helical coiled coil

Abstract

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 α-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 ∼55-60% α-helical in physiological aqueous solution at 25°C, and ∼85-90% α-helical at 4°C. From these results, we conclude that the stalk of kinesin heavy chain forms an α-helical coiled coil structure. The temperature dependence of the circular dichroism signal has two major transitions, at 25-30°C and at 45-50°C, which suggests that a portion of the α-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°C lies within coil 1, while the majority of coil 2 melts above 45 °C. We suggest that this difference in stability may play a role in the force-gene rating mechanism or regulation of kinesin.