Kinetic Characterization of the Weak Binding States of Myosin V

Abstract

Myosin V is a molecular motor shown to move processively along actin filaments. We investigated the properties of the weak binding states of monomeric myosin V containing a single IQ domain (MV 1IQ) to determine if the affinities of these states are increased as compared to conventional myosin. Further, using a combination of non-hydrolyzable nucleotide analogues and mutations that block ATP hydrolysis, we sought to probe the states that are populated during ATP-induced dissociation of actomyosin. MV 1IQ binds actin with a K_d = 4 μM in the presence of ATPγS at 50 mM KCl, which is 10−20-fold tighter than that of nonprocessive class II myosins. Mutations within the switch II region trapped MV 1IQ in two distinct M.ATP states with very different actin binding affinities (K_d = 0.2 and 2 μM). Actin binding may change the conformation of the switch II region, suggesting that elements of the nucleotide binding pocket will be in a different conformation when bound to actin than is seen in any of the myosin crystal structures to date.