ABSTRACT Tropomodulins (Tmods) are tropomyosin (TM) binding proteins that bind to the pointed end of actin filaments and modulate thin filament dynamics. They bind to the N termini of both "long" TMs (with the N terminus encoded by exon la of the alpha-TM gene) and "short" nonmuscle TMs (with the N terminus encoded by exon 1b). In this present study, circular dichroism was used to study the interaction of two designed chimeric proteins, AcTM1 aZip and AcTM1 blip, containing the N terminus of a long or a short TM, respectively, with protein fragments containing residues 1 to 130 of erythrocyte or skeletal muscle Tmod. The binding of either TMZip causes similar conformational changes in both Tmod fragments promoting increases in both a-helix and (3-structure, although they differ in binding affinity. The circular dichroism changes in the Tmod upon binding and modeling of the Tmod sequences suggest that the interface between TM and Tmod includes a three- or four-stranded coiled coil. An intact coiled coil at the N terminus of the TMs is essential for Tmod binding, as modifications that disrupt the N-terminal helix, such as removal of the N-terminal acetyl group from AcTM1aZip or striated muscle alpha-TM, or introduction of a mutation that causes nemaline myopathy, Met-8-Arg, into AcTM1aZip destroyed Tmod binding.
The actin filament network immediately under the plasma membrane in regions of cellular protrusion consists of a dendritic network of short, branched filaments. In contrast, the actin filaments deeper in the cortex, in stress fibers and microvilli, as well as those in muscle sarcomeres are much longer and rarely branched (Small, 1995; Bailly et al., 1999; Svitkina and Borisy, 1999). Actin binding proteins regulate actin filament dynamics spatially and temporally by affecting nucleation of polymerization, the kinetics of monomer addition and loss at the filament ends and the severing of the filament.
Factors that stabilize new filaments by protecting them from pointed end depolymerization or from severing can result in stable, unbranched filaments. For example, tropomyosin (TM), a coiled-coil protein that binds along the sides of actin filaments, inhibits the rate of depolymerization from the pointed end, without affecting elongation (Broschat et al., 1989; Broschat, 1990). Elongation of TM-actin filaments from the pointed end can be blocked by tropomodulin (Tmod), an actin filament pointed end-capping protein that also binds TM (Weber et al., 1994, 1999). In striated muscle, Tmod is specifically associated with actin and TM at the thin filament pointed ends where it regulates thin filament length in vivo (Gregorio et al., 1995; Gregorio and Fowler, 1995; Sussman et al., 1998; Littlefield et al., 2001). Tropomyosin also inhibits the ability of actin filaments to act as secondary activators of nucleation in the formation of filament branches by the activated Arp 2/3 complex (Blanchoin et al., 2001) and protects actin filaments from severing proteins such as actin-depolymerizing factor/cofilin (Bamburg et al., 1999).
Tropomyosin and tropomodulin both belong to multigene families that are expressed widely in eukaryotic cells. The TMs belong to two major classes: "long" ~285 residue TMs, which are expressed in muscle and nonmuscle cells and "short" ~247 residue isoforms, which are found in nonmuscle cells. In the alpha-TM gene (human TPMI) alternate promotor selection results in gene products that express exon la and 2 (residues 1-80) in long isoforms or exon lb (residues 1-43) in short isoforms (Helfman et al., 1986; Lin et al., 1988). Long and short TM isoforms share common functions including actin binding, regulation with myosin, stabilization and stiffening of the actin filament (for reviews, see Tobacman, 1996; Lin et al., 1997), and inhibition of the Arp 2/3 complex nucleated branching (Blanchoin et al., 2001), although there are some isoform differences (Novy et al., 1993; Fanning et al., 1994; Moraczewska et al., 1999). The exon la encoded sequence is highly conserved throughout phylogeny and is essential for actin binding of long TM isoforms (Cho et al., 1990; Moraczewska et al., 2000). Modifications such as lack of N-terminal acetylation or introduction of a nemaline myopathy-causing mutation, Met-8-Arg, severely reduce the actin affinity of skeletal muscle a-TM (Heald and Hitchcock-DeGregori, 1988; Urbancikova and Hitchcock-DeGregori, 1994).
Tropomodulin isoforms are also widely expressed (Fowler and Conley, 1999). Tropomodulin was originally isolated from human erythrocytes as a tropomyosin-binding protein (Fowler, 1987, 1990). Two major tropomodulin isoforms are E-tropomodulin (E-Tmod, Tmodl) and Sk-tropomodulin (Sk-Tmod, Tmod4) (Almenar-Queralt et al., 1999; Cox and Zoghbi, 2000; Conley et al., 2001). E- and Sk-Tmods differ in their tissue distribution: in mammals, E-Tmod is the predominant isoform in heart and slow skeletal muscle as well as in erythrocytes, whereas Sk-Tmod is exclusively expressed in skeletal muscle (Conley et al., 2001). In chickens, Sk-Tmod is the predominant isoform in fast skeletal muscle and in erythrocytes, whereas E-Tmod is predominant in the heart and slow skeletal muscle fibers. In chicken skeletal muscle fibers that coexpress both Sk- and E-Tmods, the Tmods are recruited to different actin filament-containing cytoskeletal structures within the cell: myofibrils and costameres, respectively (Almenar-Queralt et al., 1999). These results suggest that vertebrates have different Tmod isoforms that play distinct roles in vivo.
The Tmods have distinct TM and actin binding domains (Babcock and Fowler, 1994; Gregorio et al., 1995) with the binding site for TM being in the N-terminal half of Tmod. Tmod binds to the N terminus of TM (Sung and Lin, 1994). Recently mutagenesis studies mapped the binding for recombinant human E-Tmod to the first heptad of the coiled-coil region (residues 6-13) of human tropomyosin hTM5, a product of the TPM4 gene (Vera et al., 2000).
In this present study, circular dichroism (CD) was used to study the structural changes that accompany Tmod-TM interaction and the specificity of the interaction among different isoforms of TM and Tmod. We used recombinant constructs of full-length E-Tmod and N-terminal fragments of E- and Sk-Tmods (E-Tmod^sub 1-130^ and Sk-Tmod^sub 1-30^) that contain the TM binding domain and measured their interactions with AcTM1aZip and AcTM1bZip, two designed chimeric proteins previously used for structural studies of the N terminus of TM (Greenfield et al., 1998, 2001). AcTM 1 aZip and AcTM 1 blip contain the first 14 and 19 residues of long and short rat a-TMs encoded by exon la and exon 1b, respectively, and the 18 C-terminal residues of the leucine zipper domain of the yeast transcription factor, GCN4 (Landschulz et al., 1988). The TMZip chimeras, although very short, exhibit many of the functions of full length TMs. For example, AcTM1aZip binds to peptides containing the C terminus of striated TM to form a 1:1 complex. This binary complex binds tightly to a protein fragment containing the TM binding domain of Troponin T (TnT) to form a ternary complex with 1:1:1 stoichiometry (Palm et al., 2001).
We found that the TMZip chimeric proteins bind to full length E-Tmod and to fragments containing the N-terminal 130 residues of both Sk- and E-Tmod and that there are isoform specific differences in binding affinity. In addition, our results give insight into the structure of the Tmod-TM complexes.
MATERIALS AND METHODS
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Norma J. Greenfield* and Velia M. Fowlert
*University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635 USA; and the Scripps Research Institute, La Jolla, California 92037 USA
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