Insights into the Mechanism of Myosin V Processivity from Point Mutations in the Converter Domain

Abstract

Dimeric myosin V is an intracellular transporter, which moves cargoes towards the barbed end of actin filaments. Its ability to perform as individual molecules implies that the ATPase cycles in two head domains are coordinated, ensuring the efficient unidirectional motility. Directional loads were shown to modulate the kinetics of nucleotide binding to myosin V, suggesting that the head-head communication may be achieved via intramolecular load, generated when both heads are bound to actin. Here we directly tested the effect of the intramolecular load on the processive stepping of myosin V, using point mutations in the converter domain. The converter is a compact structure, which transmits tiny conformational changes at the nucleotide-binding site to the lever arm. To disturb the transmission mechanism, we replaced with alanines, one at a time, two phenylalanine residues that form a hydrophobic cluster with the C-terminus of the relay helix. These mutations are inferred to reduce intramolecular load but affect neither the nucleotide binding nor actin affinity. We used the combination of bulk kinetic and single-molecule measurements to study in detail the effect of the mutations in the converter on the myosin V performance. The F697A mutation, which completely eliminates intramolecular load, abolishes the motility of myosin V dimers. At the same time, the F749A mutation, which only partly reduces intramolecular load, significantly increases the proportion of backward steps. The obtained results provide strong experimental evidence that the efficient unidirectional processive stepping of myosin V is ensured by the head-head communication based on the intramolecular load.