title: A periodic pattern of evolutionarily conserved basic and acidic residues constitutes the binding interface of actin-tropomyosin.
abstract: Actin filament cytoskeletal and muscle functions are regulated by actin binding proteins using a variety of mechanisms. A universal actin filament regulator is the protein tropomyosin, which binds end-to-end along the length of the filament. The actin-tropomyosin filament structure is unknown, but there are atomic models in different regulatory states based on electron microscopy reconstructions, computational modeling of actin-tropomyosin, and docking of atomic resolution structures of tropomyosin to actin filament models. Here, we have tested models of the actin-tropomyosin interface in the "closed state" where tropomyosin binds to actin in the absence of myosin or troponin. Using mutagenesis coupled with functional analyses, we determined residues of actin and tropomyosin required for complex formation. The sites of mutations in tropomyosin were based on an evolutionary analysis and revealed a pattern of basic and acidic residues in the first halves of the periodic repeats (periods) in tropomyosin. In periods P1, P4, and P6, basic residues are most important for actin affinity, in contrast to periods P2, P3, P5, and P7, where both basic and acidic residues or predominantly acidic residues contribute to actin affinity. Hydrophobic interactions were found to be relatively less important for actin binding. We mutated actin residues in subdomains 1 and 3 (Asp(25)-Glu(334)-Lys(326)-Lys(328)) that are poised to make electrostatic interactions with the residues in the repeating motif on tropomyosin in the models. Tropomyosin failed to bind mutant actin filaments. Our mutagenesis studies provide the first experimental support for the atomic models of the actin-tropomyosin interface.
authors: Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854; Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854. Electronic address: baruabi@umdnj.edu.
authors: Bipasha
authors: B
authors: Barua
authors: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405.
authors: Patricia M
authors: PM
authors: Fagnant
authors: Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
authors: Donald A
authors: DA
authors: Winkelmann
authors: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405.
authors: Kathleen M
authors: KM
authors: Trybus
authors: Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854; Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854.
authors: Sarah E
authors: SE
authors: Hitchcock-DeGregori
mesh_terms: D000199:Actins / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002942:Circular Dichroism; D003599:Cytoskeleton / Q000378:metabolism; D019143:Evolution, Molecular; D006801:Humans; D007313:Insecta; D008854:Microscopy, Electron / Q000379:methods; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D051381:Rats; D017422:Sequence Analysis, DNA; D055672:Static Electricity; D013499:Surface Properties; D014335:Tropomyosin / Q000737:chemistry*
chemical_list: D000199:Actins; D014335:Tropomyosin