Mutational analysis of the role of hydrophobic residues in the 338-348 helix on actin in actomyosin interactions.
Yeast actin mutants with alanines replacing I341 and I345 were studied to assess the role of hydrophobic residues in the alpha-helix 338-348 in interactions with myosin. In structural models of the actomyosin complex, this helix on actin was assigned a prominent role in the strong binding of myosin to actin. Substitution of I341 with alanine reduced the strong binding of actin to myosin subfragment-1 (S1) 9-fold compared to wild-type actin. In addition, the Vmax of the actin-activated S1 ATPase was reduced 4-fold with no change in the Km. In contrast, substitution of I345 with alanine had no significant effect on either the strong binding to S1 or the actin activation of S1 ATPase. The I341A actin filaments were found to slide in the in vitro motility assays at a lower mean velocity (1.6 +/- 0.4 microns/s) than wild-type actin filaments (2.6 +/- 0.3 microns/s). Only 65% of the mutant actin filaments moved in such assays in comparison to 95% of the wild-type filaments. However, addition of 2.0 mM MgADP to the motility assay buffer induced movement of all the I341A filaments at a velocity (1.6 +/- 0.1 microns/s) similar to that of wild-type actin (1.7 +/- 0.1 microns/s). The decrease in motility of the I341A actin filaments in the absence of ADP was attributed to a negative load slowing the mutant filaments and the smaller force produced by the heavy meromyosin and I341A actin system. The latter conclusion was confirmed by showing that a greater percentage of NEM-modified heavy meromyosin (external load) was required for arresting the motion of wild-type actin in the in vitro motility assay than that needed for stopping the I341A filaments.
Miller
C J
CJ
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Doyle
T C
TC
Bobkova
E
E
Botstein
D
D
Reisler
E
E
D008841:Actin Cytoskeleton / Q000502:physiology; D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000205:Actomyosin / Q000737:chemistry / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001483:Base Sequence; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010590:Phalloidine / Q000494:pharmacology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D014364:Tryptophan / Q000378:metabolism; D015003:Yeasts / Q000737:chemistry* / Q000235:genetics
D000199:Actins; D015879:Myosin Subfragments; D010590:Phalloidine; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins