A posttranslational modification of the mitotic kinesin Eg5 that enhances its mechanochemical coupling and alters its mitotic function

Abstract

Numerous posttranslational modifications have been described in kinesins, but their consequences on motor mechanics are largely unknown. We investigated one of these-acetylation of lysine 146 in Eg5-by creating an acetylation mimetic lysine to glutamine substitution (K146Q). Lysine 146 is located in the α2 helix of the motor domain, where it makes an ionic bond with aspartate 91 on the neighboring α1 helix. Molecular dynamics simulations predict that disrupting this bond enhances catalytic site-neck linker coupling. We tested this using structural kinetics and single-molecule mechanics and found that the K146Q mutation increases motor performance under load and coupling of the neck linker to catalytic site. These changes convert Eg5 from a motor that dissociates from the microtubule at low load into one that is more tightly coupled and dissociation resistant-features shared by kinesin 1. These features combined with the increased propensity to stall predict that the K146Q Eg5 acetylation mimetic should act in the cell as a "brake" that slows spindle pole separation, and we have confirmed this by expressing this modified motor in mitotically active cells. Thus, our results illustrate how a posttranslational modification of a kinesin can be used to fine tune motor behavior to meet specific physiological needs.

Kif15 functions as an active mechanical ratchet

“…Kif15, like Eg5, Kif15, has a short run length and tends to detach from the MT before reaching stall ( Reinemann et al , 2017 ). But Eg5 is directionally insensitive—its low detachment rate under both hindering and assisting loads allows it to restrict the sliding apart of overlapping MTs ( Shimamoto et al , 2015 ) so that it both drives microtubule sliding and acts as a drag brake ( Saunders et al , 2007 ; Shimamoto et al , 2015 ; Muretta et al , 2018 ) that limits the rate of anaphase spindle elongation. By contrast, Kif15 is an active ratchet, stepping slowly and securely under hindering load, and slipping under assisting load.…”

Pathogenic Mutations in the Kinesin-3 Motor KIF1A Diminish Force Generation and Movement Through Allosteric Mechanisms

“…We thank NVIDIA for their gift of GPU card through their Academic GPU seed grant. Trajectory analyses were carried out in R using the Bio3D v2.3-3 package (84).Residue-residue distance differences between wildtype (WT) and mutant ATP-bound kinesin motor domain in complex with tubulin heterodimer were identified with ensemble difference distance matrix (eDDM) analysis routine(36,85). For this analysis, a total of 400 conformations were obtained for each state under comparison by extracting 100 equally timespaced conformations from the last 20 ns of each simulation replicate.…”

Non‐canonical functions of the mitotic kinesin Eg5

“…Accumulating evidence indicates that Eg5 localization, motor activity, and function are modulated by post‐translational modifications (Fig ) . For example, the threonine at position 926 in the tail domain of human Eg5 can be phosphorylated by Cdk1; this modification is important for the interaction of Eg5 with microtubules and its localization to the spindle .…”

“…In contrast to the Eg5 tail domain, it is unclear whether the motor domain of Eg5 is phosphorylated, and whether such phosphorylation is involved in the modulation of Eg5 localization, motor activity, or function in spindle assembly and maintenance. Recent studies have suggested acetylation of Eg5 at lysine 146, which is located in the α2 helix of the motor domain, enhances its mechanochemical coupling, and alters its mitotic function . In addition, Src is reported to phosphorylate three tyrosines in the motor domain of human Eg5 however, the molecular mechanisms regulating this modification and its functional significance remain unknown.…”