Interaction of F-actin with synthetic peptides spanning the loop region of human cardiac beta-myosin heavy chain containing Arg403

The atomic model of the F-actin-myosin subfragment 1 complex (acto-S-l) from skeletal muscle suggests that the transition of the complex from a weakly to a strongly binding state, generating mechanical force during the contractile cycle, may involve the attachment of the upper 50-kDa subdomain of myosin subfragment 1 (S-1) to the interface between subdomains 1 and 3 of actin. For the human cardiac myosin, this putative interaction would take place at the ordered loop including Arg403 of the beta-heavy chain sequence, a residue whose mutation into Gin is known to elicit a severe hypertrophic cardiomyopathy caused by a decrease of the rate of the actomyosin ATPase activity. Moreover, in several nonmuscle myosins the replacement of a Glu residue within the homolog loop by Ser or Thr also results in the reduction of the actomyosin ATPase rate that is alleviated by phosphorylation. As an approach to the characterization of the unknown interaction properties of F-actin with this particular S-l loop region, we have synthesized four 17-residue peptides corresponding to the sequence Gly398-Gly414 of the human beta-cardiac myosin. Three peptides included Arg403 (GG17) or Gln403 (GG17Q) or Ser409 (GG17S) and the fourth peptide (GG17sc) was a scrambled version of the normal GG17 sequence. Using fluorescence polarization, cosedimentation analyses and photocross-linking, we show that the three former peptides, but not the scrambled sequence, directly associate in solution to F-actin, at a nearly physiological ionic strength, with almost identical affinities (K-d approximate to 40 mu M) The binding strength of the F-actin-GG17 peptide complex was increased fivefold (K-d = 8 mu M) in the presence of subsaturating concentrations of added skeletal S-l relative to actin, without apparent competition between the peptide and S-1. Each of the three actin-binding peptides inhibited the steady-state actin-activated MgATPase of skeletal S-l by specifically decreasing about twofold the V-max of the reaction without changing the actin affinity for the S-1-ATP intermediate. Cosedimentation assays indicated the binding of about 0.65 mol peptide/mol actin under conditions inducing 70% inhibition. Collectively, the data point to a specific and stoichiometric interaction of the peptides with F-actin that uncouples its binding to S-l from ATP hydrolysis, probably by interfering with the proper attachment of the S-l loop segment to the interdomain connection of actin.