A Spacer between the Head and Neck Coil of Kinesin-1 Relieves Inhibition Due to Crosslinking of the Heads

Abstract

Kinesin-1 is autoinhibited through crosslinking of its two motor domains (heads) by a tail domain (Kaan, et al., Science 333, 883 (2011)), in addition to crosslinking of the heads by attachment to the neck coil (NC). This 'double lockdown' would prevent undocking of the neck linker (NL) and inhibit ADP release. Inhibition by double lockdown was supported the ability of a disulfide crosslink in a S181C mutant to mimic inhibition in the absence of tails. Insertion of a flexible spacer at the junction of the NL and NC could potentially provide enough 'slack' for NL undocking and ADP release. To test this mechanism, a five amino acid spacer was introduced into the S181C mutant (a total of ten additional amino acids in the dimeric construct). At 2 μM MTs, the rate of ADP release was 0.65 s−1 and <0.01 s−1 for the crosslinked forms with or without the spacer, versus 4.5 s−1 when uncrosslinked. Thus ten amino acids of slack per dimer greatly accelerates ADP release, albeit not fully up to the uncrosslinked level. That this spacer is sufficient to largely relieve the inhibition suggests that the inability to undock the NL is the principal cause of the inhibition produced by double lockdown. Studies with longer and shorter spacers will better define the dependence of the ADP release rate on the amount of slack.The assistance of James Hopkins in cloning and protein isolation is acknowledged. Supported by NIH Grant NS058848.