A Structural Model of Actomyosin in the Prepowerstroke State: Actin Binding Induced Extra Priming of Myosin

Abstract

The effective step of myosin's working cycle is the powerstroke. Although, there are atomic structural models of the actomyosin complex in rigor state, there is a great demand for atomic models of the complex in the prepowerstroke state as well, in order to understand the mechanism of the powerstroke. As the actin bound prepowerstroke state of myosin is experimentally challenging to capture because of its short life-time and low proportion of the cycle, first we created an in silico structural model of such state. We docked the relaxed structures of myosin in the weak actin binding and up lever state to an actin trimer, then relaxed the complex. Interestingly, actin binding induced a further up movement of the lever accompanied with a further closure of the switch II loop in a nanosecond time-scale. This state may represent an ‘extra primed' state of myosin that precedes the powerstroke. Additionally, when the interaction between activation loop and actin N-terminal region was interrupted in the docked and relaxed actomyosin by applying a mutation, myosin relaxed back to its original up lever conformation. In order to test our in silico results, we experimentally examined the lever movement upon actin binding. GFP and ReAsH were introduced into the C- and N-terminal of Dictyostelium myosin motor domain to follow the lever position by FRET. We conclude that in silico and experimental results reveal a new state of actomyosin at the beginning of the powerstroke, indicating the initial mechanism of actin activation of myosin.