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 fro m a weakly to a strongly binding state, generating mechanical force du ring 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 putati ve interaction would take place at the ordered loop including Arg403 o f the beta-heavy chain sequence, a residue whose mutation into Gin is known to elicit a severe hypertrophic cardiomyopathy caused by a decre ase of the rate of the actomyosin ATPase activity. Moreover, in severa l nonmuscle myosins the replacement of a Glu residue within the homolo g loop by Ser or Thr also results in the reduction of the actomyosin A TPase rate that is alleviated by phosphorylation. As an approach to th e characterization of the unknown interaction properties of F-actin wi th this particular S-l loop region, we have synthesized four 17-residu e peptides corresponding to the sequence Gly398-Gly414 of the human be ta-cardiac myosin. Three peptides included Arg403 (GG17) or Gln403 (GG 17Q) 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 thr ee former peptides, but not the scrambled sequence, directly associate in solution to F-actin, at a nearly physiological ionic strength, wit h 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 inhibite d the steady-state actin-activated MgATPase of skeletal S-l by specifi cally decreasing about twofold the V-max of the reaction without chang ing the actin affinity for the S-1-ATP intermediate. Cosedimentation a ssays 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 t hat uncouples its binding to S-l from ATP hydrolysis, probably by inte rfering with the proper attachment of the S-l loop segment to the inte rdomain connection of actin.