MAPPING MYOSIN-BINDING SITES ON ACTIN PROBED BY PEPTIDES THAT INHIBITACTOMYOSIN INTERACTION

A 2-kDa peptide (2K peptide) which was derived from the neck region of porcine aorta smooth muscle myosin heavy chain binds to actin competi tively with skeletal myosin subfragment 1 (S1) in the absence of ATP a nd inhibits acto-S1 ATPase activity [Katoh, T. and Morita, F. (1993) J . Biol. Chem. 268, 2380-2388]. Using this and other peptides, myosin-b inding sites on actin were mapped and their functions were studied, Th e 2K peptide inhibited the acto-S1 ATPase activity without inhibiting the binding of SI to actin in the presence of ATP, On the other hand, the dansylated 2K peptide (DNS-2K peptide) inhibited not only the acto -S1 ATPase activity but also the binding of S1 to actin in the presenc e of ATP, Thee, DNS-2K peptide was crosslinked to actin with 1-ethyl-3 [3-(dimethylamino)propyl] carbodiimide. Amino acid composition and seq uencing analyses of the fluorescent lysylendopeptidase-peptides of the crosslinked product indicated that DNS-2K peptide was crosslinked to acidic residues within residues 1-18 (Asp1, Glu2, Asp3, Glu4, and/or A sp11), 19-50 (Asp25), and 85-113 (Glu99 or Glu100) of actin. A competi tion experiment for the crosslinking with unlabeled 2K peptide showed that the crosslinking to residues 85-113 of actin was specific for DNS -2K peptide. In addition, isolated actin peptide 85-113 was found to s how the competitive inhibition of actin-activated ATPase activity of S 1 with respect to actin. These results suggest that the site within re sidues 1-28 of actin participates in the actin-activation of myosin AT Pase activity, and the site within residues 85-113 of actin participat es in the weak: binding of myosin to actin in the presence of ATP.