Role of residues 311/312 in actin-tropomyosin interaction. In vitro motility study using yeast actin mutant e311a/r312a.
According to the Lorenz et al. (Lorenz, M., Poole, K. J., Popp, D., Rosenbaum, G., and Holmes, K. C. (1995) J. Mol. Biol. 246, 108-119) atomic model of the actin-tropomyosin complex, actin residue Asp-311 (Glu-311 in yeast) is predicted to have a high binding energy contribution to actin-tropomyosin binding. Using the yeast actin mutant E311A/R312A in the in vitro motility assays, we have investigated the role of these residues in such interactions. Wild type (wt) yeast actin, like skeletal alpha-actin, is fully regulated when complexed with tropomyosin (Tm) and troponin (Tn). Structure-function comparisons of the wt and E311A/R312A actins show no significant differences between them, and the unregulated F-actins slide at similar speeds in the in vitro motility assay. However, in the presence of Tm and Tn, the mutation increases both the sliding speed and the number of moving filaments at high pCa values, shifting the speed-pCa curve nearly 0.5 pCa units to the left. Tm alone (no Tn) inhibits the motilities of both actins at low heavy meromyosin densities but potentiates only the motility of the mutant actin at high heavy meromyosin densities. Actin-Tm binding measurements indicate no significant difference between wt and E311A/R312A actin in Tm binding. These results implicate allosteric effects in the regulation of actomyosin function by tropomyosin.
Gerson
J H
JH
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Bobkova
E
E
Homsher
E
E
Reisler
E
E
D000199:Actins / Q000737:chemistry* / Q000235:genetics; D000494:Allosteric Regulation; D002118:Calcium / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014335:Tropomyosin / Q000737:chemistry*; D014336:Troponin / Q000378:metabolism; D015003:Yeasts
D000199:Actins; D015879:Myosin Subfragments; D014335:Tropomyosin; D014336:Troponin; D009218:Myosins; D002118:Calcium