Tropomyosin Position on F-Actin Revealed by EM Reconstruction and Computational Chemistry

• Published in: Biophysical journal 2011-02, Vol.100 (4), p.1005-1013
• Main Authors: Li, Xiaochuan (Edward), Tobacman, Larry S., Mun, Ji Young, Craig, Roger, Fischer, Stefan, Lehman, William
• Format: Article
• Language: English
• Subjects: Actins - chemistry; Actins - metabolism; Actins - ultrastructure; Amino acids; Amino Acids - metabolism; Animals; Binding sites; Computer Simulation; Density; Diffraction; Electron microscopes; Electron microscopy; Filaments; Image Processing, Computer-Assisted - methods; Imaging, Three-Dimensional; Mathematical models; Microscopy, Electron - methods; Models, Molecular; Molecules; Muscle, Motility, and Motor Proteins; Protein Binding; Proteins; Rabbits; Reconstruction; Reproducibility of Results; Searching; Static Electricity; Tapering; Tropomyosin - chemistry; Tropomyosin - metabolism; Tropomyosin - ultrastructure
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2010.12.3697

Electron microscopy and fiber diffraction studies of reconstituted F-actin-tropomyosin filaments reveal the azimuthal position of end-to-end linked tropomyosin molecules on the surface of actin. However, the longitudinal z-position of tropomyosin along F-actin is still uncertain. Without this information, atomic models of F-actin-tropomyosin filaments, free of constraints imposed by troponin or other actin-binding proteins, cannot be formulated, and thus optimal interfacial contacts between actin and tropomyosin remain unknown. Here, a computational search assessing electrostatic interactions for multiple azimuthal locations, z-positions, and pseudo-rotations of tropomyosin on F-actin was performed. The information gleaned was used to localize tropomyosin on F-actin, yielding an atomic model characterized by protein-protein contacts that primarily involve clusters of basic amino acids on actin subdomains 1 and 3 juxtaposed against acidic residues on the successive quasi-repeating units of tropomyosin. A virtually identical model generated by docking F-actin and tropomyosin atomic structures into electron microscopy reconstructions of F-actin-tropomyosin validated the above solution. Here, the z-position of tropomyosin alongside F-actin was defined by matching the seven broad and narrow motifs that typify tropomyosin's twisting superhelical coiled-coil to the wide and tapering tropomyosin densities seen in surface views of F-actin-tropomyosin reconstructions. The functional implications of the F-actin-tropomyosin models determined in this work are discussed.