Structural analysis of Candida albicans Kinesin-8: Unravelling the functional significance of the dimerization interface
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Authors
Serrano Arevalo, Jesus Danilo
Date
2025-06-04
Type
thesis
Language
eng
Keyword
Kinesin-8
Alternative Title
Abstract
Movement is a fundamental feature of life, and eukaryotic cells rely on three major motor protein families—dynein, myosin, and kinesin—to generate force and facilitate motility at both cellular and subcellular levels. Kinesins comprise 14 distinct families that interact with microtubules to regulate intracellular trafficking and chromosome segregation during mitosis. Among these, kinesin-8s are unique in exhibiting dual functionality: they not only move processively along microtubules as dimers but also remove terminal αβ-tubulin subunits from microtubule ends. These activities are critical for maintaining proper mitotic spindle dynamics and chromosome alignment and are driven by conformational changes within the motor domain coupled to ATP hydrolysis. Although the motor domain has been extensively studied, the function of the non-motor tail domain in kinesin-8 remains poorly understood, particularly with respect to its role in dimerization and motor regulation. Recent structural studies using Candida albicans Kip3 (CaKip3) as a kinesin-8 model revealed that its proximal tail forms an unusual eight-helix bundle for dimerization, contrasting with the extended coiled-coil architecture typical of other kinesins. This work investigates how the proximal tail contributes to kinesin-8 regulation by comparing the biochemical and structural properties of a near full-length CaKip3 dimer to monomeric motor domain constructs. We found that the CaKip3 dimer exhibited reduced tubulin-stimulated ATP turnover and asymmetric tubulin binding, where only one motor domain engaged with a tubulin subunit. Structural modelling with AlphaFold3 showed that in the presence of tubulin, CaKip3 adopts a compact conformation mediated by interactions between the motor domain and proximal tail. This folding is enabled by the flexible neck extension, which bends to bring these domains into proximity. Small-angle X-ray scattering (SAXS) further supports this self-folding behaviour in solution. We propose that this self-folding mechanism may represent an auto-inhibited state that regulates kinesin-8 activity. To further explore this hypothesis, we employed computational mutagenesis and nucleotide-dependent biochemical assays. The results of these studies indicate that kinesin-8 tails are modular and structurally dynamic, and that they form intramolecular contacts to limit the catalytic activity of the motor.