The Shaker-1 Mouse Myosin VIIa has a Drastically Compromised ATP Hydrolysis Mechanism

Abstract

Myosin V is a dimeric molecular motor, which transports organelles toward the barbed end of actin filaments in cells. Its highly efficient unidirectional motility requires the coordination of ATPase cycles in two head domains, to ensure that the rate-limiting ADP release almost exclusively occurs in the trailing head and the motor steps forward. Single-molecule measurements revealed that the directional loads modulate the kinetics of nucleotide binding to myosin V, suggesting that the head-head communication may be based on 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, which are inferred to reduce intramolecular load but do not affect the nucleotide binding or actin affinity. The converter is a compact structure, which transmits tiny conformational changes, induced at the nucleotide-binding site in the process of ATP hydrolysis, 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. The effects of the mutations on the myosin's V motility were tested by multiple kinetic and single-molecule assays. We found that the F749A mutation significantly increases the proportion of backward steps, whereas the F697A mutation completely abolishes the processive stepping of myosin V. These 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, which coordinates ATPase cycles in two motor domains.