The effect of the S14A mutation on the conformation and thermostability of Saccharomyces cerevisiae G-actin and its interaction with adenine nucleotides.
The actin Ser14 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 Ser14 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 Ser14 hydroxyl forms a polar bridge between the ATP gamma-phosphate and the amide nitrogen of Gly74, thus conferring additional stability on the actin small domain.
Chen
X
X
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1104, USA.
Peng
J
J
Pedram
M
M
Swenson
C A
CA
Rubenstein
P A
PA
D000199:Actins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D004355:Drug Stability; D006358:Hot Temperature; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008961:Models, Structural; D018482:Muscle, Skeletal / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012694:Serine; D013816:Thermodynamics
D000199:Actins; D012694:Serine; D000255:Adenosine Triphosphate; D008274:Magnesium; D000409:Alanine; D002118:Calcium