Neck-Linker Length is a Critical Determinant of Kinesin Processivity

Abstract

The kinesin neck-linker domain is a key mechanical element underlying processive kinesin motility. Not only is neck-linker docking thought to be the dominant conformational change in the kinesin hydrolysis cycle, chemomechanical communication between the two head domains must necessarily be transmitted through the two neck-linker domains and their shared coil-coil. Hence, the length of the neck-linker is expected to have a strong influence on kinesin run length, a quantitative measure of processivity. Across different kinesin families, motors with longer neck-linker domains, such as Kinesin-2 are generally less processive than Kinesin-1, which has the shortest neck-linker domain among N-terminal kinesins. However, there is disagreement in the literature as to whether artificially extending the Kinesin-1 neck-linker alters the motor run length. Using single molecule TIRF analysis to visualize GFP-tagged motors in 80 mM PIPES buffer, we find that lengthening the Kinesin-1 neck-linker by three amino acids results in a five-fold reduction in run length. Consistent with this, when the Kinesin-2 neck linker was matched to the effective length of Kinesin-1 by deleting three residues and substituting an alanine for a proline, the Kinesin-2 run length nearly matched that of Kinesin-1. These results demonstrate that run length scales with neck linker length for both Kinesin-1 and Kinesin-2 and is sufficient to account for differences in processivity. In addition, we find that adding positive charge to neck linker inserts enhances processivity, providing a possible explanation for the lack of dependence of run length on neck-linker length observed by others. Our data is consistent with the hypothesis that increasing neck linker compliance reduces processivity by disrupting front head gating and potentially provides a unifying principle across kinesin families - longer neck-linkers lead to less processive motors.