Molecular Dynamics Simulations Provide Further Insights into the Allosteric Regulation of the Kinesin-5 Motor Domain by Loop 5

Human kinesin-5 is a protein that oversees the proper formation of the bipolar mitotic spindle and is thus an appealing target for cancer treatment. The main group of kinesin-5 inhibitors reported to date binds to an allosteric pocket formed by loop 5 (L5), which is a key structural element believed to allosterically modulate kinesin-5 functionality. In this study, we carried out extensive molecular dynamics (MD) simulations on the motor domain of kinesin-5 in four representative catalytic states: ATP-bound, ADP-bound, without nucleotide (apo), and dually bound by ADP and the known main group inhibitor filanesib. MD trajectories were analyzed using the <i>Distance Fluctuation</i> and <i>Shortest Path Map</i> methods to compare and contrast allosteric connections across different parts of the motor domain in each of the four states. Simulations show that L5 is allosterically connected to both the nucleotide-binding site and the kinesin-5-microtubule interface. In the absence of inhibitor, L5 alternates between a "docked" conformation in the ATP and apo states and an "undocked" conformation in the ADP state. This supports the idea that the L5 binding pocket is cryptic and that inhibitor binding takes place in the ADP state. Residues Trp127 and Tyr211 were found to be crucial for the L5 conformational alternation. Once filanesib binds to the ADP form, we found that L5 stabilizes into an ATP-like conformation that prevents ADP release, possibly via sequestration of Glu118 by filanesib itself. Additionally, the presence of filanesib intensifies anomalous allosteric connections with L8, which is a crucial mediator of microtubule binding. This could explain the low affinity of kinesin-5 for the microtubule when L5 inhibitors are present. Our findings allow a deeper understanding of the key role of L5 in regulating kinesin-5 activity and how L5 inhibitors can achieve its disruption.