Examining the structure and role of the C-terminal tail domain of a motile microtubule-depolymerizing kinesin

Abstract

Kinesins are a family of force-generating enzymes that transport cellular cargos along microtubule trackways and restructure the mitotic spindle to segregate chromosomes. Members of the kinesin-8 family possess the dual ability to move along microtubules and change microtubule structure by actively removing αβ-tubulin subunits from microtubule ends. Some kinesin-8s combine these two activities to regulate the length and position of the mitotic spindle to ensure that divided nuclei reach the center of daughter cells at the end of mitosis. These activities are mainly powered by their highly conserved motor domain, but where and when they act is tightly regulated by their unique C-terminal tail domain. This thesis examined the structure and function of two distinct regions of the tail domain of kinesin-8 in the opportunistic human fungal pathogen Candida albicans (Ca). The region closest to the C-terminus, dubbed the distal tail, was found to harbour an additional microtubule binding interface that is also capable of binding curved tubulin rings and tubulin dimers using electrostatic interactions. Evidence is provided that the microtubule binding site of the distal tail overlaps, at least in part, with the motor domain. The other region of the C. albicans kinesin-8 tail, named the proximal tail, does not overtly associate with microtubules. Instead, this part of the kinesin seems to form a structure that tethers two kinesin-8 proteins together as a homodimer. The protein structure model prediction platforms, AlphaFold and AlphaFold Multimer, predict that the proximal tail folds as a nine-helix bundle that dimerizes with the help of a predicted coiled-coil forming helix that is immediately N-terminal to the proximal tail. Sedimentation velocity analysis of a purified proximal tail construct supports dimer formation in solution. Together, these findings expand our knowledge of the structural configuration of kinesin-8s and reveal more about how the unique C-terminal tail domain of kinesin-8s regulates the motile and microtubule remodelling activities of the motor domain.