X-ray crystal structure and solution fluorescence characterization of Mg.2'(3')-O-(N-methylanthraniloyl) nucleotides bound to the Dictyostelium discoideum myosin motor domain.

Mant (2'(3')-O-(N-methylanthraniloyl)) labeled nucleotides have proven to be useful tools in the study of the kinetic mechanism of the myosin ATPase by fluorescence spectroscopy. The sensitivity of the mant fluorophore to its local environment also makes it suitable to investigate the exposure of bound nucleotides to solvent from collisional quenching measurements. Here we present the crystal structure of mant-ADP and beryllium fluoride complexed with Dictyostelium discoideum myosin motor domain (S1dC) at 1.9 A resolution. We complement the structural approach with an investigation of the accessibility of the mant moiety to solvent using acrylamide quenching of fluorescence emission. In contrast to rabbit skeletal myosin subfragment 1, where the mant group is protected from acrylamide (Ksv=0.2 M-1), the fluorophore is relatively exposed when bound to Dictyostelium myosin motor domain (Ksv= 1.4 M-1). Differences between the Dictyostelium structure and that of vertebrate skeletal subfragment 1, in the region of the nucleotide binding pocket, are proposed as an explanation for the differences observed in the solvent accessibility of complexed mant-nucleotides. We conclude that protection of the mant group from acrylamide quenching does not report on overall closure of the nucleotide binding pocket but reflects more local structural changes.

[1]  Clive R. Bagshaw,et al.  Calcium regulation of molluscan myosin ATPase in the absence of actin , 1985, Nature.

[2]  I. Rayment,et al.  X-ray structures of the MgADP, MgATPgammaS, and MgAMPPNP complexes of the Dictyostelium discoideum myosin motor domain. , 1997, Biochemistry.

[3]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[4]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[5]  R. Goody,et al.  Is there a rate-limiting step before GTP cleavage by H-ras p21? , 1991, Biochemistry.

[6]  G. Matsuda,et al.  The primary structure of skeletal muscle myosin heavy chain: IV. Sequence of the rod, and the complete 1,938-residue sequence of the heavy chain. , 1991, Journal of biochemistry.

[7]  H M Holden,et al.  X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP.BeFx and MgADP.AlF4-. , 1995, Biochemistry.

[8]  C. Cremo,et al.  Interaction of myosin subfragment 1 with fluorescent ribose-modified nucleotides. A comparison of vanadate trapping and SH1-SH2 cross-linking. , 1990, Biochemistry.

[9]  D. Manstein,et al.  Kinetic characterization of the catalytic domain of Dictyostelium discoideum myosin. , 1995, Biochemistry.

[10]  D. Manstein,et al.  Dictyostelium discoideum myosin II: characterization of functional myosin motor fragments. , 1997, Biochemistry.

[11]  C A Smith,et al.  Active site comparisons highlight structural similarities between myosin and other P-loop proteins. , 1996, Biophysical journal.

[12]  Ivan Rayment,et al.  X-ray structure of the magnesium(II).ADP.vanadate complex of the Dictyostelium discoideum myosin motor domain to 1.9 A resolution. , 1996 .

[13]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[14]  M. Elzinga,et al.  Serine-324 of myosin's heavy chain is photoaffinity-labeled by 3'(2')-O-(4-benzoylbenzoyl)adenosine triphosphate. , 1989, Biochemistry.

[15]  G J Williams,et al.  The Protein Data Bank: a computer-based archival file for macromolecular structures. , 1977, Journal of molecular biology.

[16]  M. Elzinga,et al.  Amino acid sequence of rabbit skeletal muscle myosin. 50-kDa fragment of the heavy chain. , 1990, The Journal of biological chemistry.

[17]  T. Yanagida,et al.  Force-generating domain of myosin motor. , 1993, Biochemical and biophysical research communications.

[18]  R. Cooke,et al.  The mechanism of muscle contraction. , 1986, CRC critical reviews in biochemistry.

[19]  T. Hiratsuka.  Distinct structures of ATP and GTP complexes in the myosin ATPase. , 1984, Journal of biochemistry.

[20]  A. G. WEEDS,et al.  Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin , 1975, Nature.

[21]  T. Tao.  Time‐dependent fluorescence depolarization and Brownian rotational diffusion coefficients of macromolecules , 1969 .

[22]  Clive R. Bagshaw,et al.  Tryptophan fluorescence of chloramphenicol acetyltransferase: resolution of individual excited-state lifetimes by site-directed mutagenesis and multifrequency phase fluorometry. , 1995, Biochemistry.

[23]  T. Teng,et al.  Mounting of crystals for macromolecular crystallography in a free-standing thin film , 1990 .

[24]  R. Cooke,et al.  The conformation of the active site of myosin probed using mant-nucleotides. , 1995, Biophysical journal.

[25]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[26]  B. Sykes,et al.  Observation of multiple myosin subfragment 1-ADP-fluoroberyllate complexes by 19F NMR spectroscopy. , 1993, Biochemistry.

[27]  I. Rayment,et al.  X-ray structure of the magnesium(II)-pyrophosphate complex of the truncated head of Dictyostelium discoideum myosin to 2.7 A resolution. , 1995, Biochemistry.

[28]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[29]  D. Johnson,et al.  Solute accessibility to N epsilon-fluorescein isothiocyanate-lysine-23 cobra alpha-toxin bound to the acetylcholine receptor. A consideration of the effect of rotational diffusion and orientation constraints on fluorescence quenching. , 1985, Biophysical journal.

[30]  W. Kabsch.  A solution for the best rotation to relate two sets of vectors , 1976 .

[31]  A. Fisher,et al.  Structural studies of myosin:nucleotide complexes: a revised model for the molecular basis of muscle contraction. , 1995, Biophysical journal.

[32]  G. P. Reid,et al.  X-ray crystal structure analysis of the catalytic domain of the oncogene product p21H-ras complexed with caged GTP and mant dGppNHp. , 1995, Journal of molecular biology.

[33]  Brian W. Matthews,et al.  An efficient general-purpose least-squares refinement program for macromolecular structures , 1987 .

[34]  Jones Ta,et al.  Diffraction methods for biological macromolecules. Interactive computer graphics: FRODO. , 1985, Methods in enzymology.

[35]  S. Colowick,et al.  Methods in Enzymology , Vol , 1966 .

[36]  E. Reisler,et al.  Inhibition of myosin ATPase by beryllium fluoride. , 1992, Biochemistry.

[37]  D A Winkelmann,et al.  Three-dimensional structure of myosin subfragment-1: a molecular motor. , 1993, Science.

[38]  T. Hiratsuka.  New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes. , 1983, Biochimica et biophysica acta.

[39]  S. Lowey,et al.  [7] Preparation of myosin and its subfragments from rabbit skeletal muscle , 1982 .

[40]  J. Eccleston,et al.  Kinetics of the interaction of 2'(3')-O-(N-methylanthraniloyl)-ATP with myosin subfragment 1 and actomyosin subfragment 1: characterization of two acto-S1-ADP complexes. , 1991, Biochemistry.