title: Structural requirements of tropomodulin for tropomyosin binding and actin filament capping.
abstract: Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament.
authors.affiliation: Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA. kostyuas@umdnj.edu
authors.forename: Alla S
authors.initials: AS
authors.lastname: Kostyukova
authors.forename: Brian A
authors.initials: BA
authors.lastname: Rapp
authors.forename: Andy
authors.initials: A
authors.lastname: Choy
authors.forename: Norma J
authors.initials: NJ
authors.lastname: Greenfield
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Hitchcock-DeGregori
mesh_terms: D051339:Actin Depolymerizing Factors; D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D051345:Destrin; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010441:Peptide Chain Elongation, Translational / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D051348:Tropomodulin; D014335:Tropomyosin / Q000378:metabolism*
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D002352:Carrier Proteins; D051345:Destrin; D008840:Microfilament Proteins; D010446:Peptide Fragments; C495829:TMOD1 protein, human; C495832:Tmod1 protein, rat; D051348:Tropomodulin; D014335:Tropomyosin