Noncanonical interaction with microtubules via the N-terminal nonmotor domain is critical for the functions of a bidirectional kinesin

Abstract

Several kinesin-5 motors (kinesin-5s) exhibit bidirectional motility. The mechanism of such motility remains unknown. Bidirectional kinesin-5s share a long N-terminal nonmotor domain (NTnmd), absent in exclusively plus-end–directed kinesins. Here, we combined in vivo, in vitro, and cryo–electron microscopy (cryo-EM) studies to examine the impact of NTnmd mutations on the motor functions of the bidirectional kinesin-5, Cin8. We found that NTnmd deletion mutants exhibited cell viability and spindle localization defects. Using cryo-EM, we examined the structure of a microtubule (MT)-bound motor domain of Cin8, containing part of its NTnmd. Modeling and molecular dynamic simulations based on the cryo-EM map suggested that the NTnmd of Cin8 interacts with the C-terminal tail of β-tubulin. In vitro experiments on subtilisin-treated MTs confirmed this notion. Last, we showed that NTnmd mutants are defective in plus-end–directed motility in single-molecule and antiparallel MT sliding assays. These findings demonstrate that the NTnmd, common to bidirectional kinesin-5s, is critical for their bidirectional motility and intracellular functions.

Selective regulation of kinesin-5 function by β-tubulin carboxy-terminal tails

“…Release of the N-terminal cover strand interaction with the neck-linker allowed for undocking of the neck-linker from the kinesin motor domain to reset for the next step (Goulet et al, 2014). A similar density was recently observed for a structure of AMPPNP-bound Cin8, and the N-terminus of Cin8 was necessary for Cin8 plus-end motility (Singh et al, 2024). Interactions between the β-CTT and the kinesin-5 N-terminus could support these transitions during the mechanochemical cycle to promote kinesin-5 motility.…”

“…Replacing the Cin8 N-terminus with that of Kip1 results in an ~50% reduction in spindle localization (Figure 6E), phenocopying the genetic deletion of the β-CTT. This interaction between the β-CTT and Cin8 N-terminus is supported by their opposite charges and close proximity (Figure 6B,C), and recent structural work from other labs (Singh et al, 2024). Our computational isoelectric point calculations might suggest that the Cin8 and Kip1 N-termini should behave similarly (Figure 6A, Supplemental Figure 4).…”

“…The region containing loop 8 also includes β5 because loop 8 interrupts the two β5 sheets. We chose to focus on the N-terminal extension and loop 8 because both these regions have been previously indicated to play a role in kinesin-5 motility (Britto et al, 2016; Gerson-Gurwitz et al, 2011; Goulet et al, 2014; Pandey, Reithmann, et al, 2021; Singh et al, 2024). Furthermore, both regions have key differences between Cin8 and Kip1, potentially explaining their divergent behaviors: the N-terminus of Cin8 lacks two potential Cdk1 phosphorylation sites found in Kip1, and the loop 8 of Cin8 contains a large 99 residue insertion (Chee & Haase, 2010; Hoyt et al, 1992).…”