Structural Model of the Pre-Powerstroke State of the Actomyosin Complex

Abstract

We created a structural model of the ADP.Pi pre-powerstroke state of Dictyostelium myosin motor domain complexed with actin trimer by an in silico protein docking procedure followed by a long timescale molecular dynamics relaxation. Furthermore, we also modeled the ADP.Pi down lever state of myosin motor domain and the rigor complexes using 1q5q (apo structure of Dictyostelium myosin motor domain) or squid myosin S1 (3I5G) atomic structures and actin trimer model. During the molecular dynamics of the apo myosin (1q5q ) complexed with actin trimer the actin binding cleft of myosin motor domain spontaneously closed and the relaxed actomyosin rigor structure fits well with the structural rigor model determined by EM. The analysis of the ADP.Pi pre-powerstroke actomyosin complex showed that actin spontaneously induces significant conformational changes in the myosin motor domain. Most strikingly, actin further closes the closed switch 2 loop coupled with a further up movement of the lever. Interestingly, if the interaction between the N-terminal region of actin and myosin activation loop is interrupted by a single mutation (K520N) this conformational change does not occur upon actin binding. Furthermore, actin binding rearranges correlating movements of different myosin motor regions which effect was significantly reduced in the mutant. Recently we shoved experimentally that the interaction between actin and the activation loop is responsible for channeling the enzymatic pathway of actomyosin into the effective powerstroke path. The further closure of switch 2 induced by actin indicates that cocking is induced by actin mainly through the actin binding of activation loop. In order to test the indicated conformational change we have produced a myosin motor domain containg a FLASH and REASH probes located at the N and C-terminus, respectively, to sensitively follow the lever movement upon actin binding by FRET.