Intra-Motor Domain Coupling is a Strong Driver of Eg5 Motor Activity

Abstract

The tetrameric kinesin family member Eg5 walks a pair of motor domains along each of two antiparallel microtubules to help set up the mitotic spindle. Previous work has identified a strong coupling between the conformations of two structurally distant elements of the Eg5 motor domain: loop 5 (L5) and the motility-generating neck-linker. However, this work was performed in isolated monomers and how applicable these results are to higher-order assemblies of Eg5 remains unknown. Using electron paramagnetic resonance (EPR) spectroscopy, we have determined the structural relationships between the neck-linkers, L5s and nucleotide binding pockets of isolated Eg5 dimers. Intra-motor domain coupling appears to be a much stronger driver of conformation than inter-motor domain coordination_as was observed in the monomer, the major determinant of neck-linker conformation is the nucleotide state. The docking of the neck-linker upon ejection of ADP is conserved from the monomer to the dimer, and this conformational change is dependent upon the presence of an intact L5. We are currently investigating the effects of disrupting neck-linker-motor domain coupling on dimer motility. Our work forms a basis for future studies of motor domain coordination and provides context for the impact of external regulators on Eg5 activity.