Mutational analysis of Ser14 and Asp157 in the nucleotide-binding site of beta-actin

This paper compares wild-type and two mutant beta-actins, one in which Ser1 4 was replaced by a cysteine, and a second in which both Ser14, and Asp157 were exchanged (Ser14-->Cys and Ser14-->Cys,Asp157-->Ala, respectively). Bo th of these residues are part of invariant sequences in the loops, which bi nd the ATP phosphates, in the interdomain cleft of actin. The increased nuc leotide exchange rate, and the decreased thermal stability and affinity for DNase I seen with the mutant actins indicated that the mutations disturbed the interdomain coupling. Despite this, the two mutant actins retained the ir ATPase activity. In fact, the mutated actins expressed a significant ATP ase activity even in the presence of Ca2+ ions, conditions under which acti n normally has a very low ATPase activity. In the presence of Mg2+ ions, th e ATPase activity of actin was decreased slightly by the mutations. The mut ant actins polymerized as the wild-type protein in the presence of Mg2+ ion s, but slower than the wild-type in a K+/Ca2+ milieu. Profilin affected the lag phases and elongation rates during polymerization of the mutant and wi ld-type actins to the same extent, whereas at steady-state, the concentrati on of unpolymerized mutant actin appeared to be elevated. Decoration of mut ant actin filaments with myosin subfragment I appeared to be normal, as did their movement in the low-load motility assay system. Our results show tha t Ser14 and Asp157 are key residues for interdomain communication, and that hydroxyl and carboxyl groups in positions 14 and 157, respectively, are no t necessary for ATP hydrolysis in actin.