A myosin surface loop (amino acids 391-404) is postulated to be an important binding site for actin. In human beta-cardiac myosin, it contains a residue (Arg-403) that when mutated to a Glu or Trp causes hypertrophic cardiomyopathy (HCM). In Acanthamoeba, Dicytostelium and Aspergillus myosin Is and members of myosin class VI, one of the residues in this loop is invariably a Ser or a Thr. Phosphorylation of this residue is required for activity of Acanthamoeba and Dicytostelium myosin I. In almost all other myosins, this residue is either a Glu or an Asp, suggesting that a negative charge at this location is important for activity. To study the function of this loop, we have used site-directed mutagenesis and baculovirus expression of an heavy meromyosin (HMM)-like fragment of human nonmuscle myosin IIA to study mutations of this loop. The wild-type expressed HMM has similar properties to HMM prepared from nonmuscle myosin IIA isolated from human platelets. An Arg393Gln mutation (equivalent to the Arg403Gln HCM-associated mutation in human beta-cardiac muscle myosin) has essentially no effect on the actin-activated MgATPase activity or in vitro motility activity of the expressed HMM-like fragment. Three different mutations, an Asp399Lys mutation which reverses the negative charge at this site, an Asp399Ala mutation which eliminates the negative charge and a deletion mutation which removes residues 393-402 all decrease the V-max by 8-10 fold with little effect on the KATPase and decrease the rate of in vitro motility by a factor of 2-3. These data support an important role for the negative charge at this location, but show that it is not critical to enzymatic activity.
