KIF1A Repeats Cycle of ‘FREE Diffusion’ and ‘SPECIFIC Binding’ during Weak Binding State

Abstract

The nature of intermolecular interaction between motor and cytoskeletal filament during the weak binding state is not fully understood. In the case of kinesin, while structural analyses revealed that kinesin binds to a specific binding site on tubulin, motility data suggested that kinesin undergoes diffusion, searching for its next binding site. To understand how specific binding and diffusion are compatible in a single ADP state, we analyzed the motion of the single-headed kinesin KIF1A on various mutant microtubules (MTs) in the presence of ADP, using the single molecule motility assay. We prepared two series of mutant MTs. The first is a series with increased/decreased negative charges at the C-terminal tails (CTTs) of tubulin, reported to be indispensable for the weak binding of KIF1A to the MT (Okada et al., 2000). The second is a series of charged-to-alanine mutants in the H11-12 loop and H12 of tubulin (α-E415, -E416, -E418, -E421 and β-E410, -D417), found to be critical for kinesin motility and ATPase (Uchimura et al., 2010). The analyses of KIF1A movement showed that a reduction of negative charges in CTTs leads to a reduction in both the duration of interaction and the diffusion length of KIF1A, yet the diffusion constant was not greatly changed. In contrast, in most of the charged-to-alanine tubulin mutants, the diffusion constant of KIF1A increased and the duration shortened, but the diffusion length was unaffected. These results indicate that KIF1A-MT interaction in the ADP state can be modeled as an equilibrium between two substates: a dynamic ‘diffusion state’ and a static ‘binding state’. While CTTs stabilize the former, the critical residues in the H11-12 loop and H12 of tubulin stabilize the latter. This model is applicable to dimeric kinesin.