Two Conserved Lysines at the 50/20-kDa Junction of Myosin Are Necessary for Triggering Actin Activation

Abstract

Actin stimulates myosin's activity by inducing structural alterations that correlate with the transition from a weakly to a strongly bound state, during which time inorganic phosphate (P i ) is released from myosin's active site. The surface loop at the 50/20-kDa junction of myosin (loop 2) is part of the actin interface. Here we demonstrate that elimination of two highly conserved lysines at the C-terminal end of loop 2 specifically blocks the ability of heavy meromyosin to undergo a weak to strong binding transition with actin in the presence of ATP. Removal of these lysines has no effect on strong binding in the absence of nucleotide, on the rate of ADP binding or release, or on the basal ATPase activity. We further show that the 16 amino acids of loop 2 preceding the lysine-rich region are not essential for actin activation, although they do modulate myosin's affinity for actin in the presence of ATP. We conclude that interaction of the conserved lysines with acidic residues in subdomain 1 of actin either triggers a structural change or stabilizes a conformation that is necessary for actinactivated release of P i and completion of the ATPase cycle.The generation of force and movement by the myosin-actin interaction results from an ATP-driven cycle that alternates between dissociated states, weakly bound actomyosin states, and strongly bound actomyosin complexes. This cyclic interaction between actin and myosin is thought to involve the steps illustrated in Scheme 1, where A is actin and M is myosin.

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

“…During activation, we characterized the elementary steps, and concluded that there is no large reapportionment of cross-bridges among the six states (Scheme 1), indicating that an increase in the N-terminal negative charge enhances tension generated by each cross-bridge (Lu et al 2005). This result is consistent with the hypothesis that Nterminal negative charges affect strong interaction between actin and myosin molecules, presumably by changing the actomyosin interface (Cook et al 1993;Joel et al 2001). When rabbit skeletal actin (4 N-terminal negative charges) was used, tension increased further to 77%, indicating that there are still significant functional differences between yeast and rabbit actins, although they have 87% sequence identity.…”

Unidirectional Brownian motion observed in an in silico single molecule experiment of an actomyosin motor

“…3G), are shown to be involved in the actin-myosin electrostatic interaction. Loop 2 of myosin, on the other hand, has been thought to interact with actin, and the charge neutralizing mutation at the conserved lysines in loop 2 was shown to impair the myosin's ability to move an actin filament (32). The in silico charge neutralizing mutation introduced into the lysines in loop 2 (K640/K641/K642) showed that these residues are actually involved in the actin-myosin electrostatic interaction (Fig.…”

Conformationally Trapping the Actin-binding Cleft of Myosin with a Bifunctional Spin Label

“…Extensive experimental analysis has sought to define the biochemical and structural properties of W and S states of the actomyosin complex. The W state appears to be stabilized by hydrophobic interactions between loop 2 of the L50 domain of myosin and actin (13,15,48,49). K d for actomyosin weak binding using the Dicty myosin II motor domain has been previously reported as ϳ60 M (50).…”