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

Abstract

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 This paper compares wild-type b-actin with a mutant in which R177 has been replaced by aspartic acid. The mutant b-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-b-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 (A cc ) of the mutant actin was increased 10-fold as compared to wild-type actin. Filaments formed from the R177D mutant b-actin bound phalloidin.

Functional Consequences of a Mutation in an Expressed Human α-Cardiac Actin at a Site Implicated in Familial Hypertrophic Cardiomyopathy

“…Stability of Expressed Actins-To assess if the point mutation affected the intrinsic stability of the actin monomer, a DNase-I inhibition assay was performed to measure the thermal unfolding of actin, an assay that gives results similar to CD spectropolarimetry (26). When DNase-I is bound to monomeric G-actin, its enzymatic activity is inhibited.…”

“…Heatdenatured actin loses its ability to bind and inhibit the endonuclease activity of DNase-I. Note that actin denaturation is irreversible, and thus we are not measuring a thermodynamic equilibrium process, as discussed in detail in a previous study (26).…”

Selectivity of protein carbonylation in the apoptotic response to oxidative stress associated with photodynamic therapy: a cell biochemical and proteomic investigation

“…Interestingly, the Nterminal ATP domain of Hsp70 is similar to that of actin in skeletal muscle despite the low sequence similarity and the analogous positions of arginine 177 of actin and lysine 71 of Hsp, suggesting that the two proteins might utilize a similar mechanism for ATP hydrolysis. 49 This is supported by the abolition of ATPase activity by ADP -ribosylation of R177 by bacterial toxins; moreover, the lysine (K71) was found to be an essential catalytic residue for nucleotide hydrolysis by the 44-kDa N-terminal ATP domain of Hsp70. These structural relationships suggest that the carbonylation can occur on side chains of these critical residues (arginine 177 of actin and lysine 71 of Hsc71).…”

The other side of the coin: Functional and structural versatility of ADF/cofilins

“…The bound nucleotide (ATP, ADP-Pi, or ADP) in complex with a divalent cation (physiologically it is thought to be Mg 2+ ) is buried in the structure of actin. The actin monomer has a very low ATPase activity (k hydrolysis = 0.6 h -1 , for bovine thymus Mg 2+ -/ actin isoforms) (Schüler et al, 2006;Schüler et al, 2000). However, this activity is highly enhanced when actin monomers assemble into actin filaments (Carlier et al, 1984;Pantaloni et al, 1985;Pollard and Weeds, 1984).…”