Allosteric Tuning of Myosin 5a Motor Activity

Abstract

Myosin 5a is a processive vesicle transporter capable of taking multiple steps without detachment from actin. Its translocation activity, which powers cargo transport to micrometer distances, requires a range of biochemical adaptations. In this study we engineered the activity of myosin 5a by introducing mutations into two key regions of the motor domain. G227 is located at the entrance of the nucleotide binding pocket. This position is occupied by Gly only in highly processive vertebrate myosin 5a and 5b isoforms, whereas all other myosin 5 isoforms and myosins from other classes possess larger amino acids at this position. Our results show that the G227A mutation in myosin 5a causes a change in the rate-limiting step, which is ADP release in the wild type enzyme. In the mutant, a structural change taking place after ATP hydrolysis and before ADP release becomes rate limiting. The ADP release rate constant is much higher than that of the steady-state ATPase activity. Surprisingly, however, the mutant displays even higher steady-state actin attachment ratio than wild-type myosin 5a. The other region mutated in this study is the interface between the N-terminal and converter subdomains. In myosin 2, a repulsive interaction in this interface (K84-R704 in Dictyostelium myosin 2) exerts a kinetic tuning effect during the hydrolytic cycle, as determined in earlier studies. In wild-type myosin 5a this repulsive interaction is absent as the positive charge is missing at the position homologous to K84 (I67 in mouse myosin 5a). The introduction of a repulsive interaction by the I67K replacement results in a rate-limiting structural transition preceding the ATP-induced dissociation of myosin heads from actin. Thus, both studied mutations cause marked changes in the steady-state distribution of myosin structural states, which in turn alter the mechanochemical output of myosin 5a.