Converter Mutation Disrupts Lever arm Rotation in Myosin V

Abstract

Myosins are proposed to utilize a conserved structural mechanism to generate force in which small conformational changes in the active site result in a large swing of the lever arm or light chain binding region. The converter domain is a flexible region that provides a link between the catalytic domain and lever arm and is proposed to play a critical role in the allosteric communication between these two domains. We introduced the R712G mutation in the converter domain and examined the impact of this mutation on the structural and functional properties of myosin V. The mutation resulted in a 16% reduction in the maximum actin-activated ATPase rate and no change in the actin concentration at which the ATPase activity is one-half maximal (KATPase). The sliding velocities examined in the in vitro motility assay were very similar between WT and R712G MV. We have developed a novel FRET system in myosin V (MV) that allows examination of the dynamics of lever arm motion. We labeled MV 11IQ containing an N-terminal (NT) tetracysteine motif with the bisarsenical dye FlAsH (MV.NT.FlAsH). The first IQ motif of MV.NT.FlAsH was exchanged with QSY-9 labeled CaM, a non-fluorescent acceptor. We followed the motion of the lever-arm during the ATP binding (recovery stroke) and actin-activated product release (power stroke) steps using stopped-flow FRET. The R712G mutation reduced the rate of the recovery stroke by 23% while having little impact on the fast power stroke that occurs prior to phosphate release. Thus, a mutation in the converter domain can specifically impact the recovery stroke without altering the power stroke demonstrating different allosteric mechanisms are responsible for these two key structural transitions.