Glycine 699 is pivotal for the motor activity of skeletal muscle myosin.

Kinose, Fumi; Wang, S X; Kidambi, Usha; Moncman, Carole L.; Winkelmann, Donald A.

doi:10.1083/jcb.134.4.895

Cited by 89 publications

(100 citation statements)

References 45 publications

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“…In myosin, the highly conserved Gly 699 was shown to be a pivot vital to functionality by using the Gly-Ala substitution where alanine inhibits swiveling by the steric clash of its side chain with the peptide backbone (62). When the peptide backbone serves as a line of communication between distant sites, intervening highly conserved glycines are logical targets for Gly-Ala mutagenesis.…”

Section: Discussionmentioning

confidence: 99%

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

Ajtai

Garamszegi

Watanabe

et al. 2004

Journal of Biological Chemistry

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The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on ␤-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in ␤-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using sitedirected mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.The motor protein myosin is an ATPase and an actin-binding protein transducing ATP free energy into work. In muscle, myosin, actin, and ATP constitute the unitary work producer. The cyclical interaction of these components, a sequence of states each characterized as a static relation between the proteins and an intermediate in the degradation of ATP, is a contraction cycle. In a contraction cycle, myosin hydrolyzes ATP in its active site and forms a weak association with actin at a separate actin binding site. Release of phosphate from the active site initiates strong binding to actin and a large conformation change in myosin that produces work in the form of actin translation against a load. Several solved crystal structures represent intermediates in ATP hydrolysis and define myosin conformation changes in the absence of actin (1-6). Presently, we lack a detailed understanding of actomyosin structure and in particular how the elements making up the actomyosin interface contribute to mutual affinity, actin regulation of phosphate release, and the ability of myosin-bound nucleotide to modulate actomyosin affinity.Myosin consists of a globular head domain containing the enzymatic portion of the molecule and a tail involved in filament assembly. The globular head separated from its tail portion is called subfragment 1 (S1). 1 S1 interacts with filamentous actin (F-actin) consisting of polymerized actin monomers. An atomic model for F-actin built from monomeric actin crystal structures (7-9) satisfies x-ray diffraction data restraints (10) and is consistent with electron microscopic imagery (11). In...

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Section: Discussionmentioning

confidence: 99%

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

Ajtai

Garamszegi

Watanabe

et al. 2004

Journal of Biological Chemistry

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“…Like some T. gondii myosins, the Drosophila myosin IA has an Asn (Strom Morgan et al, 1994). Another striking feature of all apicomplexan myosins is the presence of a serine instead of the highly conserved glycine corresponding to Gly 699 in the chicken myosin II (Kinose et al, 1996). This structural feature has only been reported for a plant myosin (HaMyok3; D. Menzel, unpublished data) and a Caenorhabditis elegans class XII myosin (Baker and Titus, 1997).…”

Section: A Family Of Apicomplexan Myosins Presenting Divergent Structmentioning

confidence: 96%

“…The conserved glycine residue likely plays an essential role in the conformational changes around the actin-and nucleotide-binding sites, maybe functioning as a pivot point for the lever arm. Mutation of this glycine into an alanine residue dramatically alters the motor activity of the skeletal muscle myosin and the Dictyostelium discoideum myosin II, inhibiting the velocity of actin filament movement by 100-fold (Kinose et al, 1996;Patterson and Spudich, 1996;Patterson et al, 1997). Finally, class XIV myosins do not carry classical IQ motifs for the binding of light chains but harbor a conserved stretch of amino acids that might represent a very divergent form of this motif.…”

Section: A Family Of Apicomplexan Myosins Presenting Divergent Structmentioning

confidence: 99%

A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization

Hettmann

Herm

Geiter

et al. 2000

MBoC

135

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Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent structural features, TgM-A purified from parasites bound actin in an ATP-dependent manner. Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane association. Recombinant TgM-D also had a peripheral although not as sharply defined localization. Truncation of their respective tail domains abolished peripheral localization and tight membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was sufficient to confer plasma membrane localization and sedimentability. The peripheral localization of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous plasma membrane receptor and the implications for the invasion process.

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“…In addition, this relay helix bends about a hinge near its center during the pre-power stroke conformation, allowing the 50 kDa subdomain to approach closely to the N-terminal subdomain. The converter moves about this hinge while simultaneously rotating about two pivots (G695 and G706), located on either side of the SH1 helix (10,29,30). The precise locations of the glycine pivots, which may differ in different isoforms, are important in determining both the range of motion of the converter and the conserved contacts it can make with the relay.…”

Section: ) (B) the Equilibrium Equation (In Vitro)mentioning

confidence: 99%

Crystallographic findings on the internally uncoupled and near-rigor states of myosin: Further insights into the mechanics of the motor

Himmel¹,

Gourinath²,

Reshetnikova³

et al. 2002

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

110

108

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Here we report a 2.3-Å crystal structure of scallop myosin S1 complexed with ADP⅐BeF x, as well as three additional structures (at 2.8 -3.8 Å resolution) for this S1 complexed with ATP analogs, some of which are cross-linked by para-phenyl dimaleimide, a short intramolecular cross-linker. In all cases, the complexes are characterized by an unwound SH1 helix first seen in an unusual 2.5-Å scallop myosin-MgADP structure and described as corresponding to a previously unrecognized actin-detached internally uncoupled state. The unwinding of the SH1 helix effectively uncouples the converter͞lever arm module from the motor and allows crosslinking by para-phenyl dimaleimide, which has been shown to occur only in weak actin-binding states of the molecule. Mutations near the metastable SH1 helix that disable the motor can be accounted for by viewing this structural element as a clutch controlling the transmission of torque to the lever arm. We have also determined a 3.2-Å nucleotide-free structure of scallop myosin S1, which suggests that in the near-rigor state there are two conformations in the switch I loop, depending on whether nucleotide is present. Analysis of the subdomain motions in the weak actin-binding states revealed by x-ray crystallography, together with recent electron microscopic results, clarify the mechanical roles of the parts of the motor in the course of the contractile cycle and suggest how strong binding to actin triggers both the power stroke and product release.T he myosin head (subfragment one, or S1) consists of a motor domain (MD) and a lever arm that amplifies small conformational changes of the motor (Fig. 1). The MD comprises four subdomains (the 50-kDa upper and lower subdomains, the N-terminal subdomain, and the converter) linked by three flexible single-stranded joints (switch II, the relay, and the so-called SH1 helix). Motor function results from coupled rigid body rearrangements of the subdomains coordinated with conformational changes in the joints (1, 2). During the acto-myosin contractile cycle (3-5), the MD undergoes a number of conformational changes as the myosin head transduces ATP hydrolysis into mechanical work. The affinity of myosin for actin in the cycle is often described in terms of ''strong'' and ''weak'' actin-binding states. Myosin crystal structures have thus far been obtained only in the absence of actin, and three conformations have been identified, but their place in the contractile cycle has been controversial (6). The first or near-rigor conformation (7), was obtained in the absence of nucleotide, and, because the lever arm orientation was similar to that observed in electron microscopy (EM) images of actomyosin in the rigor state, this conformation was thought to be similar to a true rigor (i.e., strong actin-binding, nucleotide-free) state. More recent studies (8, 9) have shown, however, that this conformation corresponds to a weak actin-binding state occurring shortly after myosin detaches from actin, and that this state can bind to but cannot hydrolyze ATP. I...

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Glycine 699 is pivotal for the motor activity of skeletal muscle myosin.

Cited by 89 publications

References 45 publications

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

The Myosin Cardiac Loop Participates Functionally in the Actomyosin Interaction

A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization

Crystallographic findings on the internally uncoupled and near-rigor states of myosin: Further insights into the mechanics of the motor

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