Allostery Wiring Map for Kinesin Energy Transduction and Its Evolution

Abstract

How signals between the kinesin active and cytoskeletal binding sites are transmitted is an open question and an allosteric question. By extracting correlated evolutionary changes within 700+ sequences, we built a model of residues that are energetically coupled and that define molecular routes for signal transmission. Typically, these coupled residues are located at multiple distal sites and thus are predicted to form a complex, non-linear network that wires together different functional sites in the protein. Of note, our model connected the site for ATP hydrolysis with sites that ultimately utilize its free energy, such as the microtubule-binding site, drug-binding loop 5, and necklinker. To confirm the calculated energetic connectivity between non-adjacent residues, double-mutant cycle analysis was conducted with 22 kinesin mutants. There was a direct correlation between thermodynamic coupling in experiment and evolutionarily derived energetic coupling. We conclude that energy transduction is coordinated by multiple distal sites in the protein rather than only being relayed through adjacent residues. Moreover, this allosteric map forecasts how energetic orchestration gives rise to different nanomotor behaviors within the superfamily.

Small molecule allosteric uncoupling of microtubule depolymerase activity from motility in human Kinesin-5 during mitotic spindle assembly

“…Briefly, the Eg5 motor domain was expressed in BL21 (DE3) cell lines (Invitrogen) and purified by cation exchange chromatography 23 . The Eg5 motor domain basal ATPase rates were 0.209 ± 0.030 (n = 10) and its microtubule-stimulated activity was 6.615 ± 1.098 (n = 9), consistent with literature reports 17,57–59 . The human Eg5-513 plasmid, a gift from Susan Gilbert (Rensselaer Polytechnic Institute) 33 was transformed into BL21(DE3)RIL cells (New England Biolabs), and E .…”

The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry

“…Our structural and biophysical characterisations of the MKLP2 motor domain suggest that this protein and its relatives could act as an organiser or a tension sensor, rather than a classical transport motor, within the MT bundles at the spindle midzone. A non-transport role for kinesin-6s was also implied by recent computational analysis of the kinesin superfamily ( Richard et al, 2016 ). Nevertheless, MKLP2 inhibitor studies highlight the importance of the ATPase activity of MKLP2 for its role within the spindle, suggesting that the nucleotide-dependent conformational changes we have described are important for its mitotic function ( Labrière et al, 2016 ).…”

Altered chemomechanical coupling causes impaired motility of the kinesin-4 motors KIF27 and KIF7

“…Our analysis used kinesin sequences trimmed to the core motor domain, similar to the analysis of Wickstead et al (2010) , whereas a previous phylogenetic analysis that included sequences of each motor’s neck region placed Ce KLP19 as most similar to KIF4 and Dm KLP3A ( Powers et al, 2004 ). In addition, a recent study used a maximum likelihood coestimating algorithm that performs multiple sequence alignment (MSA) and phylogeny calculations in tandem and placed both Ce KLP19 and Dm KLP3A in the KIF4 subfamily ( Richard et al, 2016 ).…”