Kinesin-5/Cut7 C-terminal tail phosphorylation influence on motor regulation through multi-scale molecular modeling.

Kinesin-5 motor proteins play a vital role in mitotic spindle formation by generating essential forces during cell division that are necessary for proper chromosome segregation. Previous studies have confirmed the fundamental role of direct binding interactions between the tail and motor domains in kinesin-5-driven microtubule sliding. Post-translational modifications have emerged as an effective strategy for regulating the activity and structure of kinesin-5 motor proteins. Tail phosphorylation at nine mitotic residues has been suggested as a key regulatory mechanism for kinesin-5. For the first time to our knowledge, this study computationally examined the conformational dynamics of the unphosphorylated and phosphorylated tails of the kinesin-5 protein as they interact with the motor domains, using multi-scale molecular dynamics simulations. Fully atomistic molecular dynamics simulations of kinesin-5 homotetramers were conducted to obtain a stable full-tetramer conformation and thereby identify their interactions with the motor domain under mechanical stress. Steered molecular dynamics simulations were used to investigate the effects of post-translational modifications on the mechanical response of kinesin-5. Simulating the full assembly of kinesin-5 as it interacts with microtubule surfaces is computationally demanding. Therefore, coarse-graining was applied to reduce computational cost while maintaining accuracy. However, the phosphorylation residue parameters are not natively included in the Martini 3 force field. Thus, Martini 3 was extended to include phosphorylated serine and threonine, enabling accurate coarse-grained simulations. This study evaluates the performance of the developed parameters using coarse-grained steered molecular dynamics and extends the analysis to full tetramers embedded on microtubule surfaces, each comprising 12 tubulin subunits. These results indicate that tail phosphorylation regulates motor function by remodeling the interaction network.