The time of strong actomyosin binding depends on electrostatic interactions within the force generating region in human cardiac myosin

Abstract

Two single mutations, R694N and E45Q, were introduced in the beta isoform of human cardiac myosin to remove permanent salt bridges E45:R694 and E98:R694 in the force-generating region of myosin head. Beta isoform-specific bridges E45:R694 and E98:R694 were discovered in the molecular dynamics simulations of the alpha and beta myosin isoforms. Alpha and beta isoforms exhibit different kinetics, ADP dissociates slower from actomyosin containing beta myosin isoform, therefore, beta myosin stays strongly bound to actin longer. We hypothesize that the electrostatic interactions in the force-generating region modulate affinity of ADP to actomyosin, and therefore, the time of the strong actomyosin binding. Wild type and the mutants of the myosin head construct (1-843 amino acid residues) were expressed in differentiated C2C12 cells, and duration of the strongly bound state of actomyosin was characterized using transient kinetics spectrophotometry. All myosin constructs exhibited a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. Mutant R694N showed faster rate of ADP release from actomyosin, compared to the wild type and the E45Q mutant, thus confirming that electrostatic interactions within the force-generating region of human cardiac myosin regulate ADP release and the duration of the strongly bound state of actomyosin.