Nucleotide-induced closure of the ATP-binding pocket in myosin subfragment-1.

Hiratsuka, Toshiaki

doi:10.1016/s0021-9258(18)46976-1

Cited by 20 publications

(22 citation statements)

References 38 publications

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“…If it is assumed that S1 binding to the immobilized phenyl groups involves access to a hydrophobic crevice, then the decreased S1 binding, during steady-state MgATP hydrolysis (when S1 is in the S1**MgADP‚P i state), can be attributed to closure of this binding crevice (thereby limiting accessibility for the phenyl group). This explanation would be in accord with the recent observations and conclusions of Hiratsuka (1994) active site (S1**MgADP‚P i ) and exhibits weaker hydrophobicity. While the exact nature of this hydrophobic interaction remains to be elucidated, it is evident that the state of the nucleotide in the active site is a major determinant of this interaction.…”

Section: Discussionsupporting

confidence: 91%

“…The fluorescence quenching data of Franks-Skiba et al (1994) suggest that, although the nucleotide binding pocket can exist in open and closed states when the fluorescent nucleotide analogue is bound, the power stroke apparently is not related to the transition between these states. On the other hand, the fluorescence quenching data of Hiratsuka (1994) indicate that during steady-state hydrolysis the noncovalently bound dye PPBA is shielded from quenching, consistent with closure of the nucleotide-binding cleft.…”

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confidence: 94%

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Myosin Subfragment 1 Hydrophobicity Changes Associated with Different Nucleotide-Induced Conformations

Gopal

Burke

1996

Biochemistry

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Myosin subfragment 1 hydrophobicity was found to be sensitive to the occupancy and nature of bound nucleotide at its active site, as shown by changes in elution behavior of unmodified and chemically modified S1 during phenyl hydrophobic chromatography. The elution properties of S1 were unaltered by alkylation of SH1 (Cys-707) with N-ethylmaleimide or by covalent bridging between SH1 and SH2 (Cys-697) with p-phenylenedimaleimide with trapping of MgADP. Although addition of MgADP or MgATP to the elution buffers had minimal effect on the elution properties of these modified S1 species, the presence of these nucleotides was found to produce differential effects with unmodified S1. With MgADP, where S1 is in the S1** MgADP state, the elution times were decreased slightly, whereas with MgATP, where S1 is primarily in the S1** MgADP.Pi state, the elution times were significantly lowered, indicating reduced accessibility for the immobilized phenyl ligand. Stable S1 ternary complexes, formed with MgADP and various Pi analogues, showed elution times similar to that for S1 in the buffers containing MgATP. Thus, two main classes of nucleotide-induced S1 conformations can be defined according to their interaction with immobilized phenyl. These nucleotide-induced changes in S1 hydrophobicity correlate well with reported changes in radius of gyration of S1 associated with different states of the bound nucleotide [Wakabayashi, K., Tokunga, M., Kohno, I., Sugimoto, Y.; Hamanaka, T., Takezawa, Y., Wakabayashi, T., & Amemiya, Y. (1992) Science 258, 443-447], suggesting that the observed hydrophobicity interaction may be measuring accessibility of the immobilized phenyl ligand into a hydrophobic crevice, and that this crevice is closed or tightened when S1 is in the S1** MgADP.Pi state.

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Section: Discussionsupporting

confidence: 91%

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confidence: 94%

Myosin Subfragment 1 Hydrophobicity Changes Associated with Different Nucleotide-Induced Conformations

Gopal

Burke

1996

Biochemistry

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“…This explanation would be in accord with the recent result of Gopal and Burke (34), who showed using hydrophobic chromatography that the extent of hydrophobicity of S-1 is significantly altered upon addition of ATP. This work confirms the existence of such a site on S-1 which senses differences in the nucleotide-induced conformational states of S-1 (6,7,20).…”

Section: Discussionsupporting

confidence: 82%

“…It has been found from measurements using hydrophobic chromatography that the hydrophobicity of S-1 is sensitive to the conformational states of the ATP binding pocket (34). A noncovalent hydrophobic fluorescent dye bound to S-1 has also been used to detect the conformational changes in a hydrophobic pocket that are associated with the ATP hydrolysis reaction (20).…”

Section: Discussionmentioning

confidence: 99%

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Prodan Fluorescence Reflects Differences in Nucleotide-Induced Conformational States in the Myosin Head and Allows Continuous Visualization of the ATPase Reactions

Hiratsuka

1998

Biochemistry

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The noncovalent fluorescent probe 6-propionyl-2-(dimethylamino)naphthalene (prodan) binds stoichiometrically to myosin subfragment-1 (S-1) without affecting the ATPase and actin-binding properties of S-1. Neither ATP nor actin interferes with the prodan binding. Free prodan exhibits a green emission peak at 520 nm. However, the prodan bound to S-1 and the S-1.ADP complex shows blue emission peaks at 460 and 450 nm, respectively, which allow easy separation of the fluorescence contributions from the free and bound probes. In the S-1.ADP.Pi state, the blue emission peak is further shifted to 445 nm with a large (4.5-fold) fluorescence enhancement. Thus, prodan in the presence of S-1 exhibits predominantly blue fluorescence only during ATP hydrolysis, and so visualizes the ATPase reaction continuously. The initial velocities of the steady state of the Mg2+-, Ca2+-, and actin-activated ATPases can be conveniently calculated from the blue fluorescence changes. The ability of different nucleoside triphosphates (NTP) to enhance the blue fluorescence of prodan follows the order ATP > CTP > UTP > ITP > GTP. This order agrees with those of the extent of hydrophobicity near the ribose of the corresponding nucleoside diphosphates (NDP) trapped to S-1 with orthovanadate (Vi) [Hiratsuka, T. (1984) J. Biochem. (Tokyo) 96, 155-162] and the ability of different NTPs to support force production in muscle fibers [Regnier, M., et al. (1993) Biophys. J. 64, A250]. The rate of formation of the corresponding S-1.NDP.Vi complex also follows this order, whereas the NTPase rate follows the reverse order. These results indicate that nucleotide-induced changes in prodan fluorescence correspond to the nucleotide-induced conformational states of S-1. Thus, the use of prodan in studies of the myosin ATPase offers a new and promising approach not only to monitoring the ATPase reaction but also to investigating the structural changes during ATP hydrolysis.

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Switch‐1 instability at the active site decouples ATP hydrolysis from force generation in myosin II

2021

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Myosin active site elements (i.e., switch‐1) bind both ATP and a divalent metal to coordinate ATP hydrolysis. ATP hydrolysis at the active site is linked via allosteric communication to the actin polymer binding site and lever arm movement, thus coupling the free energy of ATP hydrolysis to force generation. How active site motifs are functionally linked to actin binding and the power stroke is still poorly understood. We hypothesize that destabilizing switch‐1 movement at the active site will negatively affect the tight coupling of the ATPase catalytic cycle to force production. Using a metal‐switch system, we tested the effect of interfering with switch‐1 coordination of the divalent metal cofactor on force generation. We found that while ATPase activity increased, motility was inhibited. Our results demonstrate that a single atom change that affects the switch‐1 interaction with the divalent metal directly affects actin binding and productive force generation. Even slight modification of the switch‐1 divalent metal coordination can decouple ATP hydrolysis from motility. Switch‐1 movement is therefore critical for both structural communication with the actin binding site, as well as coupling the energy of ATP hydrolysis to force generation.

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Nucleotide-induced closure of the ATP-binding pocket in myosin subfragment-1.

Cited by 20 publications

References 38 publications

Myosin Subfragment 1 Hydrophobicity Changes Associated with Different Nucleotide-Induced Conformations

Myosin Subfragment 1 Hydrophobicity Changes Associated with Different Nucleotide-Induced Conformations

Prodan Fluorescence Reflects Differences in Nucleotide-Induced Conformational States in the Myosin Head and Allows Continuous Visualization of the ATPase Reactions

Switch‐1 instability at the active site decouples ATP hydrolysis from force generation in myosin II

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