Charge-reversion mutagenesis of Dictyostelium actin to map the surface recognized by myosin during ATP-driven sliding motion.

Abstract

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...

Mutations in β‐actin: influence on polymer formation and on interactions with myosin and profilin

“…Interpreted in terms of the new model, this would mean that the initial myosin-dependent activation step is unaffected, but that the steps involving length changes in actin have been altered by the mutation. Results consonant with these ideas have also been obtained by others [20,[46][47][48][49].…”

Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

“…The acto-S1 docking model (50) suggests that Arg-95 of actin is involved in the interaction with myosin, and previous mutational studies suggest that negative charges in this region are important for myosin binding (48,(51)(52)(53). Consistent with this, R95C filaments, in which the net negative charge is increased in the region, showed greater affinity for myosin in in vitro motility assays.…”

“…Because actin is highly acidic, the charge reversal resulting from E226K mutation either weakens repulsion between filaments or attracts clusters of negative charges in neighbor filaments. The bundling of mutant actin filaments was also associated with mutations of the N-terminal acidic residues (48,49). However, we do not think that this bundling of E226K filaments was the cause of the dominant negative phenotype, as E226K filaments did not bundle in physiological salt concentrations or when copolymerized with WT actin (data not shown).…”

Two identical hydrophobic clusters are present on the same actin monomer: interaction between one myosin subfragment‐1 and two actin monomers

“…A few induced mutations in Drosophila were obtained (G368E and E316K), resulting in normal muscle structure while reducing S1 rigor binding [32], and altering the kinetics of force generation [33]. In contrast, substitution by histidine at E360/E361 and D363/D364 in Dictyostelium actin [34] (motif a2) resulted in wild-type motility and affected the kinetic steps of the ATPase cycle. When E93K mutation was induced, actin bound the myosin head in the rigor complex [35].…”

“…When E93K mutation was induced, actin bound the myosin head in the rigor complex [35]. Conversely, when amino residues E99/E100 (motif at) in Dictyostelium actin were replaced with histidine residues, the velocity of filament sliding in vitro was only 17% of that of wild-type F-actin [34].…”