Mutational Analysis of the Role of the N Terminus of Actin in Actomyosin Interactions. Comparison with Other Mutant Actins and Implications for the Cross-Bridge Cycle

Strongly dominant negative mutant actins, identified by An and Mogami (An, H. S., and Mogami, K. (1996) J. Mol. Biol. 260, 492-505), in the indirect flight muscle of Drosophila impaired its flight, even when three copies of the wild-type gene were present. Understanding how these strongly dominant negative mutant actins disrupt the function of wild-type actin would provide useful information about the molecular mechanism by which actin functions in vivo. Here, we expressed and purified six of these strongly dominant negative mutant actins in Dictyostelium and classified them into three groups based on their biochemical phenotypes. The first group, G156D, G156S, and G268D actins, showed impaired polymerization and a tendency to aggregate under conditions favoring polymerization. G63D actin of the second group was also unable to polymerize but, unlike those in the first group, remained soluble under polymerizing conditions. Kinetic analyses using G63D actin or G63D actin⅐gelsolin complexes suggested that the pointed end surface is defective, which would alter the polymerization kinetics of wild-type actin when mixed and could affect formation of thin filament structures in indirect flight muscle. The third group, R95C and E226K actins, was normal in terms of polymerization, but their motility on heavy meromyosin surfaces in the presence of tropomyosin-troponin indicated altered sensitivity to Ca 2؉ . Cofilaments in which R95C or E226K actins were copolymerized with a 3-fold excess of wild-type actin also showed altered Ca 2؉ sensitivity in the presence of tropomyosin-troponin.Actin filaments are major components of the cytoskeletons of all eukaryotic cells and play key roles in a variety of cellular functions, including cell migration, organelle transport, and muscle contraction. These diverse processes depend on the dynamic nature of actin filaments and their interactions with various actin-binding proteins (1), which are thought to involve conformational changes in the subunits that make up actin filaments. For instance, the stability of actin filaments is affected by the conformational difference between actin subunits carrying ATP or ADP and those that do not (2, 3), whereas cofilin, a major actin filament severing protein, alters the twist of actin filaments (4). In addition, modification of actin filaments through chemical cross-linking causes motility defects with myosin, suggesting a possible link between actin-myosin interaction and the conformational dynamics of actin (5, 6). Moreover, the myosin-induced conformational changes appear to be cooperative (7-9), e.g. Ca 2ϩ -triggered conformational changes in the skeletal muscle actin filaments are enhanced by cooperative conformational changes induced by myosin (10). In many cases, however, details of the molecular relationship between conformational changes in actin and its physiological function remain unclear.Actin is a highly conserved protein, so that Dictyostelium actin 15 shares 91% amino acid identity with rabbit skeletal muscle actin. This implies ...

Section: Velocitymentioning

confidence: 62%

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

Noguchi

Gomibuchi

Murakami

et al. 2010

“…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.…”

Section: Resultsmentioning

confidence: 91%

“…Our HMM mutation was more severe in that even at low ionic strength, motility was not observed. Based on mutations in actin, Reisler and colleagues (16) proposed that the interaction between acidic residues in subdomain 1 of actin and the residues in loop 2 facilitates the flux from weak to strong binding states. Here we come to similar conclusions but further show that the conserved positive residues in loop 2 of myosin are essential for triggering any flux from the weak to strong binding state.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

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

Joel¹,

Trybus²,

Sweeney³

2001

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.

“…Methylcellulose enhances solution viscosity, reducing diffusion of actin filaments away from the surface and increasing the probability of further interactions with myosin heads. It thus maintains movement at very low myosin surface densities (27) or when actomyosin affinity is reduced, either by various actin or myosin mutants (6,38) or by higher ionic strengths (39,40). Indirect flight muscle myosin from Lethocerus (waterbug) has a K m for actin, measured from actin-activated ATPase studies, which is 6 -15 times greater than vertebrate fast muscle (41).…”

Section: Discussionmentioning

confidence: 99%

Alternative Exon-encoded Regions of Drosophila Myosin Heavy Chain Modulate ATPase Rates and Actin Sliding Velocity

Swank¹,

Bartoo

Knowles

et al. 2001

137

To investigate the molecular functions of the regions encoded by alternative exons from the single Drosophila myosin heavy chain gene, we made the first kinetic measurements of two muscle myosin isoforms that differ in all alternative regions. Myosin was purified from the indirect flight muscles of wild-type and transgenic flies expressing a major embryonic isoform. The in vitro actin sliding velocity on the flight muscle isoform (6.4 m⅐s ؊1 at 22°C) is among the fastest reported for a type II myosin and was 9-fold faster than with the embryonic isoform. With smooth muscle tropomyosin bound to actin, the actin sliding velocity on the embryonic isoform increased 6-fold, whereas that on the flight muscle myosin slightly decreased. No difference in the step sizes of Drosophila and rabbit skeletal myosins were found using optical tweezers, suggesting that the slower in vitro velocity with the embryonic isoform is due to altered kinetics. Basal ATPase rates for flight muscle myosin are higher than those of embryonic and rabbit myosin. These differences explain why the embryonic myosin cannot functionally substitute in vivo for the native flight muscle isoform, and demonstrate that one or more of the five myosin heavy chain alternative exons must influence Drosophila myosin kinetics.