A conserved negatively charged amino acid modulates function in human nonmuscle myosin IIA.
A myosin surface loop (amino acids 391-404) is postulated to be an important actin binding site. In human beta-cardiac myosin, mutation of arginine-403 to a glutamine or a tryptophan causes hypertrophic cardiomyopathy. There is a phosphorylatable serine or threonine residue present on this loop in some lower eukaryotic myosin class I and myosin class VI molecules. Phosphorylation of the myosin I molecules at this site regulates their enzymatic activity. In almost all other myosins, the homologous residue is either a glutamine or an aspartate, 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 a heavy meromyosin- (HMM-) like fragment of human nonmuscle myosin IIA. An R393Q mutation (equivalent to the R403Q mutation in human beta-cardiac muscle myosin) has essentially no effect on the actin-activated MgATPase or in vitro motility of the expressed HMM-like fragment. Three mutations, D399K, D399A, and a deletion mutation that removes residues 393-402, all decrease both the V(max) of the actin-activated MgATPase by 8-10-fold and the rate of in vitro motility by a factor of 2-3. The K(ATPase) of the actin-activated MgATPase activity and the affinity constant for binding of HMM to actin in the presence of ADP are affected by less than a factor of 2. These data support an important role for the negative charge at this location but show that it is not critical to enzymatic activity.
Wang
F
F
Laboratory of Molecular Cardiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1762, USA.
Harvey
E V
EV
Conti
M A
MA
Wei
D
D
Sellers
J R
JR
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D016367:Baculoviridae; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015879:Myosin Subfragments / Q000737:chemistry / Q000235:genetics; D009218:Myosins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D055672:Static Electricity
D000199:Actins; D020409:Molecular Motor Proteins; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins