Coupling the Actin Binding Cleft and Nucleotide Binding Pocket in Myosin V

Abstract

Previously we have demonstrated in fluorescence resonance energy transfer (FRET) studies that mant labeled nucleotides and IAEDANS actin can act as good donor probes for a FlAsH labeled acceptor site in the upper 50 kDa domain of myosin V. We examined the temperature dependence of the FRET signal between mantADP and MV WT FlAsH in the presence and absence of actin. We found that at low temperature (4-15°C) a high FRET state dominates (closed pocket) while at high temperature (30-35°C) a low FRET state dominates (open pocket). This transition is reversible suggesting a temperature-dependent conformational change. However, the mutant E442A, which is incapable of hydrolyzing ATP, remains in a high FRET state (closed pocket) with mantATP bound in the presence or absence of actin. Our results suggest a more flexible conformation of myosin in the presence of ADP compared to ATP which allows myosin to populate two actomyosin.ADP state conformations. These results are supported by the lifetime FRET analysis, and by computational FIRST/FRODA analysis of the intrinsic flexibility found in different x-ray crystal structures. We also plan to explore the temperature dependent conformational dynamics of the actin binding cleft using the IAEDANS actin (donor) and MV FlAsH (acceptor) pair in the presence of ATP, ADP, and absence of nucleotide using steady state and lifetime based FRET measurements. Our results will provide critical insights into the mechanocoupling that may occur between the nucleotide-binding pocket and actin binding cleft in myosin motors.