Mutational analysis of arginine 177 in the nucleotide binding site of beta-actin.

Actin ADP-ribosylated at arginine 177 is unable to hydrolyze ATP, and the R177 side chain is in a position similar to that of the catalytically essential lysine 71 in heat shock cognate protein Hsc70, another member of the actin-fold family of proteins. Therefore, actin residue R177 has been implicated in the mechanism of ATP hydrolysis. This paper compares wild-type beta-actin with a mutant in which R177 has been replaced by aspartic acid. The mutant beta-actin was expressed in Saccharomyces cerevisiae and purified by DNase I-affinity chromatography. The mutant protein exhibited a reduced thermal stability and an increased nucleotide exchange rate, suggesting a weakened interdomain connection. The ATPase activity of G-actin and the ATPase activity expressed during polymerization were unaffected by the R177D replacement, showing that this residue is not involved in catalysis. In the presence of polymerizing salts, ATP hydrolysis by both wild-type Mg-beta-actin and the mutant protein preceded filament formation. With the mutant actin, the initial rate of ATP hydrolysis was as high as with wild-type actin, but polymer formation was slower, reached lower steady-state levels, and the polymers formed exhibited much lower viscosity. The critical concentration of polymerization (Acc) of the mutant actin was increased 10-fold as compared to wild-type actin. Filaments formed from the R177D mutant beta-actin bound phalloidin.