The interaction of Phe472 with a fluorescent inhibitor bound to the complex of myosin subfragment-1 with nucleotide.
The fluorescent probe 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonyl amido]phenylboronic acid (PPBA) acts as a fluorescent inhibitor for the ATPases of skeletal [Hiratsuka (1994) J. Biol. Chem. 269, 27251-27257] and Dictyostelium discoideum [Bobkov et al. (1997) J. Muscle Res. Cell Motil. 18, 563-571] myosins. The former paper suggested that, upon addition of excess nucleotides to the binary complex of subfragment-1 from skeletal myosin (S1) with PPBA, a stable ternary complex of S1 with PPBA and nucleotide is formed. Useful fluorescence properties of PPBA enable us to distinguish the conformation of the myosin ATPase at the ATP state from that at the ADP state. In the present paper, to determine the PPBA-binding site in the complexes, enzymatic and fluorescence properties of the S1.PPBA.nucleotide complexes were investigated. Upon formation of the ternary complex with ATP, a new peak appeared at 398 nm in the PPBA fluorescence spectrum. Experiments using model compounds of aromatic amino acid suggested that this fluorescence peak at 398 nm is originated from PPBA interacting with Phe residue(s). Taking into account differences in fluorescence spectra between complexes of S1 and those of subfragment-1 from D. discoideum myosin (S1dC), in the ternary complex of S1 formed with ATP, PPBA was suggested to interact with Phe residue(s) that is absent in S1dC. Docking simulation of PPBA on the S1.nucleotide complex revealed that Phe472 interacts with PPBA. Binding sites of PPBA and blebbistatin, an inhibitor showing high affinity and selectivity toward myosin II [Kovács et al. (2004) J. Biol. Chem. 279, 35557-35563], seem to overlap at least partly.
Hiratsuka
Toshiaki
T
The Department of Chemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan. toshiaki@asahikawa-med.ac.jp
D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites / Q000187:drug effects; D001897:Boronic Acids / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000737:chemistry / Q000494:pharmacology; D008958:Models, Molecular; D015879:Myosin Subfragments / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects*; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence / Q000379:methods; D013449:Sulfonamides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
C509418:3-(4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonylamido)phenylboronic acid; D001897:Boronic Acids; D005456:Fluorescent Dyes; D006576:Heterocyclic Compounds, 4 or More Rings; D015879:Myosin Subfragments; D009711:Nucleotides; D013449:Sulfonamides; C472645:blebbistatin; D014443:Tyrosine; D010649:Phenylalanine; D014364:Tryptophan; D000255:Adenosine Triphosphate