Amino acid residues D24/D25, E99/E100, E360/E361, and D363/E364 in subdomain 1 of Dictyostelium actin were replaced with histidine residues by site-directed mutagenesis. Mutant actins were expressed in Dictyostelium cells and purified to homogeneity. The sliding movement of mutant actin filaments on heavy meromyosin attached to a glass surface was measured to assess the effect of the mutation on the motility of actin. For two C-terminal mutants, force generated by a single actin ifiament and myosin was also measured. These measurements indicated that both D24/D25 and E99/E100 are involved in ATP-driven sliding, whereas E360/E361/ D363/E364 are not essential for ATP-driven sliding and force generation.Comparison of the atomic model of fiamentous (F) actin (1, 2) with a cryoelectron microscopic image of an actin-myosin complex formed in the absence ofMgATP (3) showed that the actin N terminus, the loop of residues 20-25, and the hydrophobic helix of residues 340-350 are in the major contact site of the myosin head (4). The helix of residues 79-93 and the following loop are also in contact with a long extension of the myosin head (4). The helix containing acidic residues E360/ E361/D363/E364 is in contact with the N-terminal segment of the alkaline light chain 1 of the myosin head (3, 5). The actin-myosin interface thus identified coincides with that identified by antibody mapping and chemical cross-linking studies (5-11). The question is, however, whether the interface identified in the absence ofMgATP is actually recognized by myosin heads during ATP-driven sliding and force generation. One approach to answer this question is to use recombinant actin (12)(13)(14)(15). It has been shown (12) that mutational disruption of the N-terminal acidic residue cluster ofDictyostelium actin affected its sliding on heavy meromyosin (HMM). The result indicates that the N-terminal acidic residues are recognized by the myosin head during sliding and, possibly, force generation.Besides the N-terminal cluster ofacidic amino acid residues, several acidic residue pairs (D24/D25, E99/E100, E360/E361, and D363/E364) are found in the putative actin-myosin interface (Fig. 1). To map the actin surface recognized by the myosin head during actin-myosin sliding and force generation, these pairs of acidic residues were replaced with histidine by recombinant DNA techniques. Since it has been wellestablished that the ionic interaction between actin and myosin is dominant during sliding and force generation, a chargereversion mutation at the actin-myosin interface would result in the loss of sliding and force generation. In fact, examination of the sliding of these mutant actin filaments by an in vitro motility assay (16,17) showed that mutations at D24/D25 and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.at E99/E100 resulted in the complete or partial loss offilament sliding...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.