Free Energy Changes During Kinesin's Force-Generating Substep

Abstract

We have previously suggested that Kinesin-1 generates force by transient folding between the N-terminal cover strand and the C-terminal neck linker domains into a beta-sheet, the so-called cover-neck bundle (CNB). Once formed, the CNB has a conformational bias sufficient to move the neck linker forward. Replica exchange molecular dynamics simulations have been performed to elucidate the energetics of CNB formation with and without load. Without load, the CNB state is weakly favorable compared to non-CNB states by 0.85 kcal/mol at 300 K, which is in agreement with a previous experimental value based on electron paramagnetic resonance, 0.72 kcal/mol (Rice et al, Biophys. J. 84:1844 (2003)), although the identity of the states involved is not certain. In non-CNB states the mobile neck linker points mostly forward in the ATP-like conformation of the motor head, so there is relatively little conformational difference with the CNB-state. By contrast, when a 10-pN rearward load is applied at the end of the beta9 part of the neck linker, a new local energy minimum appears for a rearward-pointing state. Compared to the CNB state, the free energy of the rearward-pointing state is higher by 2.96 kcal/mol at 300 K. This indicates that the CNB readily forms under applied load and thus is able to move the neck linker forward. The significance of these results for the mechanism by which kinesin-1 walks on microtubules will be discussed.