Role of the salt-bridge between switch-1 and switch-2 of Dictyostelium myosin 1 1Edited by A. R. Fersht

Furch, Marcus; Fujita‐Becker, Setsuko; Geeves, Michael A.; Holmes, Kenneth C.; Manstein, Dietmar J.

doi:10.1006/jmbi.1999.2921

Cited by 86 publications

(98 citation statements)

References 45 publications

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“…4D). Most myosin isoforms investigated to date, including smooth muscle myosin II (20), vertebrate non-muscle myosin II (17,21), Dictyostelium myosin II (22), as well as myosins I (23,24), V (25), and VI (26), show rigor dissociation constants in the nanomolar range, and the dissociation rate constants fall usually below 0.01 s Ϫ1 under similar temperature and ionic strength conditions to those in the current study. An intriguing exception is skeletal muscle myosin, which showed a very similar profile to mX-S1 (K A ϭ 0.11 M, k A ϭ 0.22 s Ϫ1 at 20°C, I ϭ 125 mM) in the pyrene fluorescence study of Criddle et al (18).…”

Section: Discussionsupporting

confidence: 63%

Mechanism of Action of Myosin X, a Membrane-associated Molecular Motor

Kovács

Wang

Sellers

2005

Journal of Biological Chemistry

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We have performed a detailed biochemical kinetic and spectroscopic study on a recombinant myosin X head construct to establish a quantitative model of the enzymatic mechanism of this membrane-bound myosin. Our model shows that during steady-state ATP hydrolysis, myosin X exhibits a duty ratio (i.e. the fraction of the cycle time spent strongly bound to actin) of around 16%, but most of the remaining myosin heads are also actin-attached even at moderate actin concentrations in the so-called "weak" actin-binding states. Contrary to the high duty ratio motors myosin V and VI, the ADP release rate constant from actomyosin X is around five times greater than the maximal steadystate ATPase activity, and the kinetic partitioning between different weak actin-binding states is a major contributor to the rate limitation of the enzymatic cycle. Two different ADP states of myosin X are populated in the absence of actin, one of which shows very similar kinetic properties to actomyosin⅐ADP. The nucleotide-free complex of myosin X with actin shows unique spectral and biochemical characteristics, indicating a special mode of actomyosin interaction.Myosin X is a recently described member of the myosin superfamily that is expressed in vertebrate tissues as a single isoform (1, 2). The heavy chain of myosin X consists of an N-terminal motor domain containing the actin-and ATP-binding sites, a neck region that binds three calmodulin (or possibly other) light chains, a putative coiled-coil region that may bring about heavy chain dimerization, and a tail region consisting of several domains of effector function (three pleckstrin homology (PH3) 1 domains, a MyTH4, and a FERM domain) (1). The presence of the PH3 domains in the myosin X tail is a unique feature among myosins, and it enables this myosin to directly bind to the plasma membrane via phosphatidylinositol phospholipids (1, 3). Myosin X has been shown to localize to regions of dynamic actin and to exhibit remarkable patterns of intrafilopodial motility (1, 4). Most interestingly, myosin X localizes to the tips of filopodia, which appears to be an active process requiring myosin X motor function (4). Myosin X induces elongation of filopodia by transporting the Mena-VASP complex (an inhibitor of actin filament capping) to filopodial tips (5). Myosin X activity is also necessary for phagocytosis (6) as well as the localization and function of integrins (7).The above functional studies indicate a cellular role for myosin X as a plasma membrane-associated cargo transporter. This setting is unique within the myosin superfamily, which implies that this class of motors may have adapted to its role by acquiring distinctive molecular properties. All myosins exert their motile activity during a cyclic interaction with actin filaments and ATP. The enzymatic parameters are key determinants of the motile output of motor proteins, and it has been shown in numerous cases that the biochemistry of different myosins reflects precise and profound functional adaptations to their widely differing c...

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Section: Discussionsupporting

confidence: 63%

Mechanism of Action of Myosin X, a Membrane-associated Molecular Motor

Kovács

Wang

Sellers

2005

Journal of Biological Chemistry

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“…8, A and B). Previously, it has been shown that this residue plays a key role in the mechanism of chemo-mechanical coupling (36) and that mutation of this residue abolishes the cellular functions of myosin-2 in D. discoideum cells (19). Accordingly, the elongation of the switch-2 region in myosin-18A by a flexible linker (GGSARGA) and the disturbed salt bridge between switch-1 and switch-2 are possible structural determinants for the absent intrinsic ATPase activity of the motor domain.…”

Section: Discussionmentioning

confidence: 99%

“…Kinetic Measurements-ATPase activities were measured at 25°C with the NADH-coupled assay as described previously (19). Transient kinetic experiments were performed at 20°C with either a Hi-tech Scientific SF-61 DX single mixing stopped-flow system (TgK Scientific Ltd., Bradford on Avon, UK) or an Applied Photophysics PiStar 180 instrument in assay buffer (20 mM MOPS (pH 7.0), 100 mM KCl, 5 mM MgCl 2 , 1 mM DTT) using procedures and kinetic models described previously (Scheme 1) (20,21).…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of Human Myosin-18A and Its Interaction with F-actin and GOLPH3

Taft

Behrmann

Munske-Weidemann

et al. 2013

Journal of Biological Chemistry

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Background: Class-18A myosins share a unique N-terminal extension comprising a PDZ module and a KE-rich region. Results: Human myosin-18A binds F-actin via its motor domain in a nucleotide-dependent manner and via the KE-rich region, modulated by direct interaction between the PDZ module and GOLPH3. Conclusion: Myosin-18A binds F-actin and recruits interaction partners to the cytoskeleton. Significance: This work establishes a molecular basis for myosin-18A mediated membrane-cytoskeleton interplay.

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“…T he mechanism of hydrolysis of adenosine triphosphate (ATP) by myosin, leading to adenosine diphosphate (ADP) and inorganic phosphate (P i ), which constitutes one of the most important enzymatic reactions responsible for energy transduction into the directed movements of adjoining actin filaments, continues to remain a subject of active debates (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), a significant part of which relates to what constitutes the acceptor of the proton that must be released by the ''lytic'' water in its nucleophilic attack on the ATP ␥-phosphate.…”

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confidence: 99%

Mechanism of the myosin catalyzed hydrolysis of ATP as rationalized by molecular modeling

Grigorenko

Rogov

Topol

et al. 2007

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

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The intrinsic chemical reaction of adenosine triphosphate (ATP) hydrolysis catalyzed by myosin is modeled by using a combined quantum mechanics and molecular mechanics (QM/MM) methodology that achieves a near ab initio representation of the entire model. Starting with coordinates derived from the heavy atoms of the crystal structure (Protein Data Bank ID code 1VOM) in which myosin is bound to the ATP analog ADP⅐VO 4 ؊ , a minimum-energy path is found for the transformation ATP ؉ H 2O 3 ADP ؉ Pi that is characterized by two distinct events: (i) a low activation-energy cleavage of the P ␥OO␤␥ bond and separation of the ␥-phosphate from ADP and (ii) the formation of the inorganic phosphate as a consequence of proton transfers mediated by two water molecules and assisted by the Glu-459 -Arg-238 salt bridge of the protein. The minimum-energy model of the enzyme-substrate complex features a stable hydrogen-bonding network in which the lytic water is positioned favorably for a nucleophilic attack of the ATP ␥-phosphate and for the transfer of a proton to stably bound second water. In addition, the P␥OO␤␥ bond has become significantly longer than in the unbound state of the ATP and thus is predisposed to cleavage. The modeled transformation is viewed as the part of the overall hydrolysis reaction occurring in the closed enzyme pocket after ATP is bound tightly to myosin and before conformational changes preceding release of inorganic phosphate.ATP hydrolysis ͉ enzymatic catalysis ͉ energy profile ͉ quantum mechanics and molecular mechanics simulations T he mechanism of hydrolysis of adenosine triphosphate (ATP) by myosin, leading to adenosine diphosphate (ADP) and inorganic phosphate (P i ), which constitutes one of the most important enzymatic reactions responsible for energy transduction into the directed movements of adjoining actin filaments, continues to remain a subject of active debates (1-16), a significant part of which relates to what constitutes the acceptor of the proton that must be released by the ''lytic'' water in its nucleophilic attack on the ATP ␥-phosphate.In terms of the generally accepted kinetic scheme (1-3), the relevant ATP-myosin transformations may be described by the equationin which M* and M** indicate conformers of myosin. As reported (1-3), reaction (Eq. 1) occurs with a near unit equilibrium constant K Ͻ 10 and the estimated rate constants k ϩ Ն 160 s Ϫ1 and k Ϫ Ն 18 s Ϫ1 . The rate constant k ϩ ϭ 160 s Ϫ1 can be converted to the free-energy activation barrier ⌬G # Ϸ 14.6 kcal/mol at room temperature, T ϭ 300 K, by applying a simple transition-state theory formula (17) k Ϸ 6⅐10 12 exp͓Ϫ⌬G # ͞RT͔. [2]However, noting that the experimental rate constants of reaction (Eq. 1) incorporate contributions from conformational changes in the protein from M* to M** leads us to expect that the activation energy of the intrinsic chemical reactionwhich excludes conformational rearrangements, should be considerably Ͻ14.6 kcal/mol. However, previous attempts (13, 15, 16) to simulate the mechanism of react...

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Role of the salt-bridge between switch-1 and switch-2 of Dictyostelium myosin 1 1Edited by A. R. Fersht

Cited by 86 publications

References 45 publications

Mechanism of Action of Myosin X, a Membrane-associated Molecular Motor

Mechanism of Action of Myosin X, a Membrane-associated Molecular Motor

Functional Characterization of Human Myosin-18A and Its Interaction with F-actin and GOLPH3

Mechanism of the myosin catalyzed hydrolysis of ATP as rationalized by molecular modeling

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