Local destabilization of the tropomyosin coiled coil gives the molecular flexibility required for actin binding.
Tropomyosin, a coiled coil protein that binds along the length of actin filaments, contains 40 uninterrupted heptapeptide repeats characteristic of coiled coils. Yet, it is flexible. Regions of tropomyosin that may be important for binding to the filament and for interacting with troponin deviate from canonical coiled coil structure in subtle ways, altering the local conformation or energetics without interrupting the coiled coil. In a region rich in interface alanines (an Ala cluster), the chains pack closer than in canonical coiled coils, and are staggered, resulting in a bend [Brown et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8496-8501]. Brown et al. suggested that bends at alanine clusters allow tropomyosin to wind on the actin filament helix. Another explanation is that local destabilization of the coiled coil, rather than close packing of the chains at Ala clusters per se, allows flexibility. Changing three Ala residues to canonical interface residues, A74L-A78V-A81L, greatly stabilized tropomyosin, measured using circular dichroism and differential scanning calorimetry, and reduced actin affinity >10-fold. Normal actin affinity and stability were restored in a mutant A74Q-A78N-A81Q that mimicked the stability of the Ala cluster but not the close packing of the chains. Analysis and modeling of comparable mutations introduced closer to the N-terminus show that the effects on stability and function depend on context. Models based on tropomyosin crystal structures give insight into possible effects of the mutations on the structure. We conclude that the significance of the Ala clusters in allowing flexibility of tropomyosin is stability-driven.
Singh
Abhishek
A
Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA. singha2@umdnj.edu
Hitchcock-DeGregori
Sarah E
SE
D000199:Actins / Q000737:chemistry*; D000409:Alanine / Q000737:chemistry / Q000235:genetics; D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D003198:Computer Simulation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D013816:Thermodynamics; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics
D000199:Actins; D014335:Tropomyosin; D000409:Alanine