Zoheb Ahmed1, Shyamalava Mazumdar2, Krishanu Ray3. 1. Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, 400005, India. 2. Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, 400005, India. 3. Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, 400005, India. Electronic address: krishanu@tifr.res.in.
Abstract
The heterotrimeric kinesin-2 consists of two distinct motor subunits and an accessory protein, KAP, which binds to the coiled-coil stalk domains and one of the tail domains of the motor subunits. Genetic studies revealed that KAP is essential for the kinesin-2 functions in cilia, flagella, and axon. However, the structural significance of the KAP binding on kinesin-2 assembly and stability is not known. Here, using the Fluorescence Lifetime assay, we show that DmKAP binding selectively reduces the distance between the C-terminal ends of Drosophila kinesin-2 stalk heterodimer. Insertion of a missense mutation (E551K) in the Drosophila kinesin-2α stalk fragment, which was shown to reduce the structural dynamics of the stalk heterodimer earlier, also reduced the distances at both the N- and C-terminal ends of the stalk heterodimer independent of DmKAP. The zipping effect, particularly at the N-terminal end of the mutant stalk heterodimer, is further enhanced in the presence of DmKAP. Together, these results suggest that the KAP binding could alter the structural dynamics of kinesin-2 stalk heterodimer at the C-terminal end and stabilize the association between the stalk domains.
The heterotrimeric kinesin-2 consists of two distinct motor subunits and an accessory protein, n class="Gene">KAP, which binds to the coiled-coil stalk domains and one of the tail domains of the motor subunits. Genetic studies revealed that KAP is essential for the kinesin-2 functions in cilia, flagella, and axon. However, the structural significance of the KAP binding on kinesin-2 assembly and stability is not known. Here, using the Fluorescence Lifetime assay, we show that DmKAP binding selectively reduces the distance between the C-terminal ends of Drosophilakinesin-2 stalk heterodimer. Insertion of a missense mutation (E551K) in the Drosophilakinesin-2α stalk fragment, which was shown to reduce the structural dynamics of the stalk heterodimer earlier, also reduced the distances at both the N- and C-terminal ends of the stalk heterodimer independent of DmKAP. The zipping effect, particularly at the N-terminal end of the mutant stalk heterodimer, is further enhanced in the presence of DmKAP. Together, these results suggest that the KAP binding could alter the structural dynamics of kinesin-2 stalk heterodimer at the C-terminal end and stabilize the association between the stalk domains.