Kinetic properties and small-molecule inhibition of human myosin-6

► First description of the production and detailed functional characterization of a human myosin-6 motor. ► Identification of halogenated phenols as inhibitors of myosin-6 activity. ► In vitro and in vivo characterization of the effect of tri-iodophenole on myosin-6 function. Myosin-6 is an actin-based motor protein that moves its cargo towards the minus-end of actin filaments. Mutations in the gene encoding the myosin-6 heavy chain and changes in the cellular abundance of the protein have been linked to hypertrophic cardiomyopathy, neurodegenerative diseases, and cancer. Here, we present a detailed kinetic characterization of the human myosin-6 motor domain, describe the effect of 2,4,6-triiodophenol on the interaction of myosin-6 with F-actin and nucleotides, and show how addition of the drug reduces the number of myosin-6-dependent vesicle fusion events at the plasma membrane during constitutive secretion.