Tension-induced suppression of allosteric conformational changes explains coordinated stepping of kinesin-1

The dimeric motor protein kinesin-1 walks along microtubules by alternating ATP hydrolysis and movement of its two motor domains (“head”). The detached head preferentially binds to the forward tubulin-binding site after ATP binding, but the mechanism preventing premature binding to the microtubule while awaiting ATP remains unknown. Here, we examined the role of the neck linker connecting two heads in this mechanism. High-resolution structural analyses of the nucleotide-free head revealed a bulge just forward of the neck linker’s base, creating an asymmetric constraint on its mobility. While the neck linker can stretch freely backward, it must navigate around the bulge to extend forward. Based on this finding, we hypothesized that the tethered head’s premature binding is suppressed due to an intolerable increase in neck linker tension. Molecular dynamic simulations and single-molecule fluorescent assays supported this model. These findings provide a universal tension-based regulation mechanism in which off-pathway conformational transitions are thermodynamically suppressed due to the entropy loss associated with neck linker stretching.