Dual requirement for flexibility and specificity for binding of the coiled-coil tropomyosin to its target, actin.
The coiled coil is a widespread motif involved in oligomerization and protein-protein interactions, but the structural requirements for binding to target proteins are poorly understood. To address this question, we measured binding of tropomyosin, the prototype coiled coil, to actin as a model system. Tropomyosin binds to the actin filament and cooperatively regulates its function. Our results support the hypothesis that coiled-coil domains that bind to other proteins are flexible. We made mutations that alter interface packing and stability as well as mutations in surface residues in a postulated actin binding site. Actin affinity, measured by cosedimentation, was correlated with coiled-coil stability and local instability and side chain flexibility, analyzed with circular dichroism and fluorescence spectroscopy. The flexibility from interruptions in the stable coiled-coil interface is essential for actin binding. The surface residues in a postulated actin binding site participate in actin binding when the coiled coil within it is poorly packed.
Singh
Abhishek
A
Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Hitchcock-DeGregori
Sarah E
SE
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D013696:Temperature; D013816:Thermodynamics; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D014335:Tropomyosin