Structural Basis for the Coordinated Processive Movement of Kinesin-1 studied by Structural and Single Molecule Analyses

Abstract

Kinesin-1 is a dimeric motor protein that moves along microtubules in a hand-over-hand manner. Recent single molecule studies uncovered how ATP hydrolysis cycle is couple to the conformational changes of kinesin dimer, although structural basis for the coordinated walking mechanism still remained unknown. At the last annual meeting, we reported the first crystal structure of nucleotide-free kinesin-1 and presented a model to explain mechanochemical coupling of kinesin motor domain. Here, we modeled atomic-detailed kinesin dimer structures on microtubule at various nucleotide states, based on docking of the crystal structures to high-resolution cryo-EM density maps (Sindelar et al. 2010) and refinement of the neck-linker structures using MD simulations. Based on the analysis of these dimer structures, we propose a model to explain the coordinated motility of kinesin-1: 1) In the two-head bound state, ATP binding to the leading head is prohibited because the neck-linker will be stretched out, explaining the front-head-gating mechanism. 2) In the one-head-bound state, ADP release from the tethered head is prohibited at the rear binding site and only permitted at the forward binding site because the neck-linker will be again stretched out, which explains the forward stepping mechanism. These models provide a testable prediction that if the neck-linker is artificially extended, kinesin can adopt these off-pathway conformations because the tension is now relaxed, and single molecule FRET analysis of neck linker extension mutants provided experimental support. These results suggest that two kinesin motor domain can coordinate to move hand-over-hand because off-pathway transitions are energetically unfavorable as they are associated with the increase in the neck linker tension.