Mutational Analysis of the Role of Hydrophobic Residues in the 338−348 Helix on Actin in Actomyosin Interactions

Miller, Cora; Doyle, Timothy C.; Bobkova, Elena; Botstein, David; Reisler, Emil

doi:10.1021/bi952645v

Cited by 39 publications

(54 citation statements)

References 33 publications

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“…This hydrophobic interaction network has not yet appeared in the weakly bound complex, so we speculate that the new formation of these hydrophobic interactions moves the triplet along the conduit of the first actin, leading finally to stabilizing the actin-myosin interaction. Our view agrees well with various proposals (7,27,28), namely that hydrophobic **Yeast actin mutants with alanine replacing Ile-341 or Ile-345 grow normally, although the mutant with alanines or lysines replacing both isoleucines does not survive (26). So we suppose that a remaining isoleucine can still support the linear sliding of the hydrophobic triplet of myosin, although the movement may not be so smooth as that obtained by a pair of isoleucines.…”

Section: Comparison Between Two Complex Modelssupporting

confidence: 91%

A closer look at energy transduction in muscle

Onishi

Morales

2007

Proc. Natl. Acad. Sci. U.S.A.

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Muscular force is the sum of unitary force interactions generated as filaments of myosins move forcibly along parallel filaments of actins, understanding that the free energy required comes from myosin-catalyzed ATP hydrolysis. Using results from conventional biochemistry, our own mutational studies, and diffraction images from others, we attempt, in molecular detail, an account of a unitary interaction, i.e., what happens after a traveling myosin head, bearing an ADP-P i, reaches the next station of an actin filament in its path. We first construct a reasonable model of the myosin head and actin regions that meet to form the ''weakly bound state''. Separately, we consider Holmes' model of the rigor state [Holmes, K. C., Angert, I., Kull, F. J., Jahn, W. & Schrö der, R. R. actin ͉ ATPase ͉ mutation ͉ myosin H ere, we attempt a molecular account of the central interactions that occur when muscle contracts or sustains tension. Because of a peculiar structural organization of muscle, the contractile apparatus can be thought of as effectively a linear filament of myosin molecules forcibly advancing along a parallel filament of actin molecules, the process being thermodynamically paid for by losing free energy of myosin-bound ATP hydrolysis (1). In this sense, contraction is an instance of transducing chemical (free) energy into mechanical work. For our purposes, however, it is simpler to consider our task to be explaining how a traveling myosin head, bearing a partly hydrolyzed ATP, on reaching interaction distance to an actin pair, fastens to it, first weakly and later strongly, and then, how it, in a remarkable cooperation with the actins, casts off the terminal phosphate of ATP and delivers a mechanical impulse to the actins.The account that follows is put together by using reports from many laboratories. These are of three general kinds, conventional biochemical [e.g., Scheme 1, § showing the timedependence of forming complexes of myosin and actin (2-4)]; mutational [e.g., recent works identifying myosin surface loops engaged in complexing with actin (5, 6)]; and x-ray diffraction [e.g., studies of Rayment (7-10), of Cohen (11), and of their pioneering associates]. Application of diffraction techniques has dramatically improved resolution and has shown that a globular head of myosin has specialized ''organs,'' like an enzyme pocket in which to conduct ATP hydrolysis, a long, stiff ␣-helix (''relay helix'') to transmit linear force, a converter to turn it into a rotation, and a lever arm to deliver a mechanical impulse to (at the time attached) actins (Fig. 1). In addition, from fitting crystal structures of actin and the myosin head to the 3D structure reconstructed from cryoEM pictures of actin filaments decorated with S1 (an isolated single myosin head), Rayment et al. (7) have suggested that ionic and hydrophobic residues, which are possibly involved in actin binding, are localized at one end of the myosin head, which is far from either the enzyme pocket or the converter (Fig. 1). At the end of the head, th...

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Section: Comparison Between Two Complex Modelssupporting

confidence: 91%

A closer look at energy transduction in muscle

Onishi

Morales

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…3B). In agreement with published comparisons (42), myosin S1-ADP had ϳ10-fold lower affinity for wild type yeast actin than for unlabeled rabbit muscle actin (Fig. 3B, squares versus circles).…”

Section: Tropomyosin Has Increased Affinity For D56a/e57a Actin Compasupporting

confidence: 91%

An Actin Subdomain 2 Mutation That Impairs Thin Filament Regulation by Troponin and Tropomyosin

Korman¹,

Hatch²,

Dixon³

et al. 2000

Journal of Biological Chemistry

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Striated muscle thin filaments adopt different quaternary structures, depending upon calcium binding to troponin and myosin binding to actin. Modification of actin subdomain 2 alters troponin-tropomyosin-mediated regulation, suggesting that this region of actin may contain important protein-protein interaction sites. We used yeast actin mutant D56A/E57A to examine this issue. The mutation increased the affinity of tropomyosin for actin 3-fold. The addition of Ca 2؉ to mutant actin filaments containing troponin-tropomyosin produced little increase in the thin filament-myosin S1 MgATPase rate. Despite this, three-dimensional reconstruction of electron microscope images of filaments in the presence of troponin and Ca 2؉ showed tropomyosin to be in a position similar to that found for muscle actin filaments, where most of the myosin binding site is exposed. Troponin-tropomyosin bound with comparable affinity to mutant and wild type actin in the absence and presence of calcium, and in the presence of myosin S1, tropomyosin bound very tightly to both types of actin. The mutation decreased actin-myosin S1 affinity 13-fold in the presence of troponin-tropomyosin and 2.6-fold in the absence of the regulatory proteins. The results suggest the importance of negatively charged actin subdomain 2 residues 56 and 57 for myosin binding to actin, for tropomyosinactin interactions, and for regulatory conformational changes in the actin-troponin-tropomyosin complex.Cardiac and skeletal muscle contraction is controlled by a complex allosteric system in which myosin-actin interactions are regulated by tropomyosin and troponin. Tropomyosin is an extended, dimeric coiled-coil and spans seven actin monomers on the thin filament. Troponin is composed of three subunits: troponin T connects the other two subunits and tropomyosin to the actin filament, troponin I inhibits myosin binding to actin, and troponin C serves as a Ca 2ϩ sensor for the system (for reviews see Refs. 1 and 2). Structural studies have shown that tropomyosin can bind to three different regions of the actin filament (3-7). In the absence of Ca 2ϩ , tropomyosin appears to bind to subdomain 1 of actin and to bridge over subdomain 2 to the next actin monomer (4). In the presence of Ca 2ϩ , tropomyosin moves toward subdomains 3 and 4 (3) and moves even further in the presence of myosin (8). Whereas these and other results support a three state model of muscle regulation involving tropomyosin movement (9 -12), information defining the position and interactions of troponin on the filament and actin residues that specifically interact with the regulatory proteins is limited.One strategy to examine muscle protein-protein interactions has been to exploit functionally altered mutants (13-15). For example, a Drosophila melanogaster actin subdomain 2 mutant, E93K, which inhibits tropomyosin-actin sliding in isolated protein preparations, has been utilized to investigate tropomyosin-based regulation (16). These and other data (17)(18)(19)(20) suggested that actin subdomain 2 may be ...

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“…Yet the sliding velocity of the N-terminal mutant actins decreased relative to that of the wild type at all levels. At higher ionic strength conditions, the sliding of the DNEQ and ⌬DSE actins ceased even in the presence of methylcellulose (44). Thus, the N-terminal actin residues contribute to acto-S1 binding in both the weakly and strongly bound states.…”

Section: Discussionmentioning

confidence: 96%

Nat3p and Mdm20p Are Required for Function of Yeast NatB Nα-terminal Acetyltransferase and of Actin and Tropomyosin

Polevoda

Cardillo

Doyle

et al. 2003

Journal of Biological Chemistry

122

139

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NatB N␣-terminal acetyltransferase of Saccharomyces cerevisiae acts cotranslationally on proteins with MetGlu-or Met-Asp-termini and subclasses of proteins with Met-Asn-and Met-Met-termini. NatB is composed of the interacting Nat3p and Mdm20p subunits, both of which are required for acetyltransferase activity. The phenotypes of nat3-⌬ and mdm20-⌬ mutants are identical or nearly the same and include the following: diminished growth at elevated temperatures and on hyperosmotic and nonfermentable media; diminished mating; defective actin cables formation; abnormal mitochondrial and vacuolar inheritance; inhibition of growth by DNAdamaging agents such as methyl methanesulfonate, bleomycin, camptothecin, and hydroxyurea; and inhibition of growth by the antimitotic drugs benomyl and thiabendazole. The similarity of these phenotypes to the phenotypes of certain act1 and tpm1 mutants suggests that such multiple defects are caused by the lack of acetylation of actin and tropomyosins. However, the lack of acetylation of other unidentified proteins conceivably could cause the same phenotypes. Furthermore, unacetylated actin and certain N-terminally altered actins have comparable defective properties in vitro, particularly actin-activated ATPase activity and sliding velocity.The two cotranslational processes, cleavage of N-terminal methionine residues and N-terminal acetylation, are by far the most common modifications, occurring on the vast majority of proteins. Eukaryotic cytosolic proteins initiate with methionine that is cleaved from nascent chains of most proteins. Subsequently, N-terminal acetylation occurs on certain of the proteins, either containing or lacking the methionine residue. This N-terminal acetylation occurs on over one-half of soluble eukaryotic proteins but seldom on prokaryotic proteins (1-3).N-terminal acetylation of proteins is catalyzed by N-terminal acetyltransferases (NATs) 1 that transfer acetyl groups from acetyl-CoA to termini of ␣-amino groups. We have established that Saccharomyces cerevisiae contains three major NATs, NatA, NatB, and NatC, with catalytic subunits Ard1p, Nat3p, and Mak3p, respectively, and that each is required for acetylating different groups of proteins (4, 5). As summarized previously (4, 5), subclasses of proteins with Ser-, Ala-, Gly-, or Thr-termini are acetylated by NatA; proteins with Met-Glu-or Met-Asp-termini and subclasses of proteins with Met-Asn-and Met-Met-termini are acetylated by NatB; and subclasses of proteins with Met-Ile, Met-Leu-, Met-Trp-, or Met-Phe-termini are acetylated by NatC. In addition, a special subclass of NatA substrates with Ser-Glu-, Ser-Asp-, Ala-Glu-, or Gly-Glu-termini, designated NatAЈ, are only partially acetylated in nat3-⌬ or mak3-⌬ mutants (6).In regard to the present study, special emphasis should be made of the NatB substrates, some of which were previously identified by using two-dimensional gel electrophoresis of proteins from normal and nat3-⌬ strains, including the following: actin (Act1p); the small subunit of ribonucleotide...

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Mutational Analysis of the Role of Hydrophobic Residues in the 338−348 Helix on Actin in Actomyosin Interactions

Cited by 39 publications

References 33 publications

A closer look at energy transduction in muscle

A closer look at energy transduction in muscle

An Actin Subdomain 2 Mutation That Impairs Thin Filament Regulation by Troponin and Tropomyosin

Nat3p and Mdm20p Are Required for Function of Yeast NatB Nα-terminal Acetyltransferase and of Actin and Tropomyosin

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