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.

FIGURE 1.

Tropomyosin mutations at conserved surface residues. The rat striated αTm sequence showing conserved b, c, and f residues that were mutated to Ala in the first half of periods P1–P7. The names of the mutants on the left indicate the basic (blue), acidic (red), or hydrophobic (gray) residues that were mutated in the first half (1) of each period (P1–P7). The pattern of basic and acidic residues at positions f, b, and f are indicated by the blue (position f, basic residues) and red (positions b and f, acidic residues) boxes.

FIGURE 2.

Actin affinity of tropomyosin mutants measured by cosedimentation with F-actin. Tropomyosin (0.1–8 μm) was combined with 5 μm chicken skeletal α-actin and sedimented at 20 °C in 200 mm NaCl, 10 mm Tris-HCl (pH 7.5), 2 mm MgCl₂, and 0.5 mm DTT. Stoichiometric binding of one Tm per seven actins is represented by fraction maximal binding of 1. The data for each mutant and WT were obtained from two to six independent experiments. The K_app values are reported in Table 1.

FIGURE 3.

Thermal stability of Tm mutants measured by circular dichroism. Fraction folded as measured by relative ellipticity at 222 nm as a function of temperature. The Tm concentration was 1.5 μm in 0.5 m NaCl, 10 mm sodium phosphate (pH 7.5), 1 mm EDTA, and 1 mm DTT. The fraction folded is relative to the mean residue ellipticity at 0 °C, where the proteins were fully folded. The T_M values are reported in Table 1.

FIGURE 4.

Model for actin-tropomyosin interaction. The model was constructed by docking a 2.3 Å Tm crystal structure (blue, Protein Data Bank code 2B9C) (21) including periods P4 and P5, and a 6.6 Å F-actin structure (green, Protein Data Bank code 3MFP) (47) as described in Barua et al. (20). The model shows potential electrostatic and hydrophobic interactions between Tm residues in P4 and P5 and actin residues. The zoomed portion shows the actin-Tm interface in P5. The model was constructed using the University of California, San Francisco Chimera package (48).

FIGURE 5.

Smooth muscle WT and D25A/E334A/K326A/K328A (mutant) α-actins. A, EM images of F-actin negatively stained with uranyl acetate. B, affinity of actins for WT Tm measured by cosedimentation assay. Tropomyosin (0.1–8 μm) was combined with 3 μm F-actin and sedimented at 20 °C in 200 mm NaCl, 10 mm Tris-HCl (pH 7.5), 2 mm MgCl₂, and 0.5 mm DTT. The K_app values are reported in Table 2. C, filament speed of actins in in vitro motility assays. The values are mean ± S.D. from two experiments. An anti-myosin subfragment 2 monoclonal antibody was bound to nitrocellulose-coated glass coverslips and then incubated with 40 μg/ml chicken skeletal myosin at 4 °C for 2 h. The coverslips were transferred to 15-μl drops of 2 nm rhodamine-phalloidin labeled smooth muscle F-actins in motility buffer (25 mm imidazole, pH 7.6, 25 mm KCl, 4 mm MgCl₂, 7.6 mm MgATP, 50 mm DTT, 0.5% methyl cellulose), and an oxygen scavenger system (0.1 mg/ml glucose oxidase, 0.02 mg/ml catalase, 2.5 mg/ml glucose) and 1–2 min of continuous video of movement of actin filaments were recorded from several fields for each experiment at 27 °C.

FIGURE 6.

Summary of contributions of basic, acidic, and hydrophobic residues in individual periodic repeats to tropomyosin function. A, tropomyosin residues at conserved surface positions that were mutated to Ala in the first half of periods P1–P7 (basic residues in blue, acidic residues in red, and hydrophobic residues in gray). The numbers indicate the reduction in actin affinity of the mutants compared with WT Tm (K_WT/K_mut). In P1, P4, and P6, primarily basic residues contribute to actin affinity (blue boxes), in contrast to P2, P3, P5, and P7, where both basic and acidic or mostly acidic residues contribute to actin affinity (red boxes). The hydrophobic residues have a smaller contribution to actin affinity. B, tropomyosin mutations shown in the 7 Å striated muscle αTm structure (Protein Data Bank code 1C1G) (49).