| Kinesins are a superfamily of proteins that transform chemical energy into directed motion along a microtubule (MT) substrate, in order to transport a specific cargo in a spatially- and temporally-regulated manner. In this work we begin to decipher the regulation of kinesin-based transport by investigating the structure of the cargo binding-domain of kinesin, known as the tail, and how its structure facilitates its interactions with various ligands.;The tail domain of human Kif5B contains an ATP-independent MT binding-site, and we investigated this interaction. The tail binds to MTs tightly, and this binding is largely due to electrostatic interactions. The MT binding-site in the tail is located at the interface between the coiled-coil stalk and the tail. The tail binding-site contains the MT E-hooks, and overlaps with the binding-site of tau, but not the binding-site of the kinesin motor. The tail is also able to promote polymerization and stability of MT filaments in a manner similar but not identical to tau.;In the absence of bound-cargo, Kif5B adopts a compact conformation in which the tail inhibits the ATPase activity of the motors. We investigated the interaction between the head and tail domains. We found that tail binding did not result in a global change in conformation of the motor domains, and our data suggested that the tail binds in the vicinity of the ATP binding-pocket. These results are described in light of a differing model for the head-tail interaction as proposed by the Hackney laboratory.;Lastly, we investigated the structure of the tail itself. A bioinformatics analysis of the tails of all kinesins suggested that most tails are intrinsically disordered (ID). These predictions were experimentally tested for four specific tails, and found to be true. A comparison of the regions of kinesin molecules that have a high predicted probability of being ID to known ligand-binding or post-translational modification sites showed a high positive correlation, suggesting that ID is a structural element that is important for the regulation and binding of ligands by kinesins. We then began preliminary investigations of the structural basis for the ligand-tail interactions for several ligands and kinesins. |
