Nonspecific Weak Actomyosin Interactions:  Relocation of Charged Residues in Subdomain 1 of Actin Does Not Alter Actomyosin Function

Abstract

Yeast actin mutants with relocated charged residues within subdomain 1 were constructed so we could investigate the functional importance of individual clusters of acidic residues in mediating actomyosin weak-binding states in the cross-bridge cycle. Past studies have established a functional role for three distinct pairs of charged residues within this region of yeast actin (D2/E4, D24/D25, and E99/E100); the loss of any one of these pairs resulted in the same impairment in weak actomyosin interaction and in its function. However, the specificity of myosin interaction with these sites has not yet been addressed. To investigate this, we made and analyzed two new actin mutants, 4Ac/D24A/D25A and 4Ac/E99A/E100A. In these mutants, the acidic residues of the D24/D25 or E99/E100 sites were replaced with uncharged residues (alanines) and a pair of acidic residues was inserted at the N-terminus, maintaining the overall charge density of subdomain 1. Using the in vitro motility assays, we found that the sliding and force generation properties of these mutant actins were identical to those of wild-type actin. Similarly, actin-activated ATPase activities of the mutant and wild-type actins were also indistinguishable. Additionally, the binding of S1 to these mutant actins in the presence of ATP was similar to that of wild-type actin. These results show that relocation of charged residues in subdomain 1 of actin does not affect the weak actomyosin interactions and actomyosin function.

Functional Consequences of a Mutation in an Expressed Human α-Cardiac Actin at a Site Implicated in Familial Hypertrophic Cardiomyopathy

“…These differences are likely a result of the fact that yeast actin has only two acidic residues at its N terminus, compared with four in cardiac actin. In agreement with this interpretation, the motility, V max , K m , as well as average force of E99A/E100A yeast actin were restored to WT values when two additional negative charges were added to the N terminus (30). Thus in yeast actin, four negative charges in subdomain 1 are more important for WT-actin function than their exact location.…”

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

“…The actin partners of the C-terminal lysine residues of loop 2 are likely in subdomain 1 of actin. The effects of removing negative charge from the N terminus, residues 24/25, or residues 99/100 of actin (15,16,36) are similar to the effects of removing two conserved positive charges from loop 2 of myosin. Mutations in either myosin or actin caused a large decrease in actin activation of ATPase activity, a more modest reduction in binding to actin in the presence of ATP, and impairments in motility.…”

Molecular mechanism of actomyosin-based motility

“…Chimaeras composed of the Dictyostelium myosin-II backbone and of loop-2 regions from myosins of other species showed actin-activated ATPase activities that correlated well with the activity of the myosins from which the loop sequences were derived [107]. Further studies showed that the initial weak binding of myosin to actin is an electrostatic interaction between positively charged lysine residues in loop 2 and negatively charged residues in subdomain 1 of actin [64,66,101,105,[108][109][110][111]. Removal of the negative charge from subdomain 1 of actin decreases the affinity of actin for myosin in the presence of Mg. ATP, while relocation of the charge in this subdomain does not alter actomyosin function, consistent with limited stereospecificity of the weak binding interaction [110,112].…”