THE EFFECT OF THE S14A MUTATION ON THE CONFORMATION AND THERMOSTABILITY OF SACCHAROMYCES-CEREVISIAE G-ACTIN AND ITS INTERACTION WITH ADENINE-NUCLEOTIDES

The actin Ser(14) hydroxyl is one of a number of ligands that binds to the gamma-phosphate of ATP thereby stabilizing the actin ATP complex. In yeast actin, conversion of Ser(14) to Ala (S14A), causes a temperature-sensitive phenotype in vivo and temperature-sensitive polymerization defects in vitro (Chen, X., and Rubenstein, P. A. (1995) J. Biol. Chem. 270, 11406-11414). Here, using a new luciferase-based procedure, we show that the mutation results in a 40-60-fold decrease in actin's affinity for ATP. The mutation causes a decrease in the intrinsic ATPase activity of both Ca- and Mg-G-actin at 30 degrees C and alters the protease susceptibility of sites on subdomain 2. Ca-S14A-actin but not Mg-S14A-actin binds etheno-ATP at 37 degrees C, Intrinsic tryptophan fluorescence measurements show that at 37 degrees C, Mg-S14A-actin but not the calcium form unfolds, CD measurements show the mutation causes a decrease in the apparent denaturation temperature for Ca-actin from 57 to 45 degrees C and for the magnesium form a decrease from 52 to 40 degrees C. Based on a re-examination of actin's crystal structure coordinates, we propose that the Ser(14) hydroxyl forms a polar bridge between the ATP gamma-phosphate and the amide nitrogen of Gly(74), thus conferring additional stability on the actin small domain.