Structural basis for capping protein sequestration by myotrophin (V-1)

Abstract

Capping protein (CP) is a ubiquitously expressed, heterodimeric 62-kDa protein that binds the barbed end of the actin filament with high affinity to block further filament elongation. Myotrophin (V-1) is a 13-kDa ankyrin repeat-containing protein that binds CP tightly, sequestering it in a totally inactive complex in vitro. Here, we elucidate the molecular interaction between CP and V-1 by NMR. Specifically, chemical shift mapping and intermolecular paramagnetic relaxation enhancement experiments reveal that the ankyrin loops of V-1, which are essential for V-1/CP interaction, bind the basic patch near the joint of the alpha tentacle of CP shown previously to drive most of the association of CP with and affinity for the barbed end. Consistently, site-directed mutagenesis of CP shows that V-1 and the strong electrostatic binding site for CP on the barbed end compete for this basic patch on CP. These results can explain how V-1 inactivates barbed end capping by CP and why V-1 is incapable of uncapping CP-capped actin filaments, the two signature biochemical activities of V-1.

FIGURE 1.

Crystal structure of chicken CPα1β1 (14). The α-subunit is shaded blue; the β-subunit is shaded yellow. N and C termini of each subunit are indicated. Each subunit contains an N-terminal 3-helix bundle followed by a peripheral loop region. The core of the protein is composed of a central β-sheet with each subunit contributing five strands. Above the central β-sheet, there are two anti-parallel α-helices, one from each subunit ending in the C-terminal tentacle. The region of each subunit thought to be flexible, based on crystallographic and mutational analysis, and thus defined as the tentacle, is highlighted in red (Arg²⁵⁹ to the C terminus in CPα and Arg²⁴⁴ to the C terminus in CPβ).

FIGURE 2.

Summary of ¹³C^α, ¹³C′ secondary chemical shifts for CP. ¹³C^α and ¹³C′ chemical shifts were compared with average random coil values for CPα (A) and CPβ (B). Positive bars correspond to the α-helical conformation; negative bars correspond to the β-sheet. Red boxes indicate flexible regions as determined by NMR T₂ relaxation studies. Secondary structures are illustrated below the shifts in blue.

FIGURE 3.

¹⁵N TROSY effective-T₂ relaxation of CP. ¹⁵N T₂ was acquired at 32 °C at 800 MHz and plotted versus residue number for CPα (A) and CPβ (B). Secondary structures are shown at the top, and numbering corresponds to that in Fig. 1.

FIGURE 4.

Chemical shift map of V-1 binding on CP. Chemical shift change was plotted against residue number for CPα (A) and CPβ (B). Secondary structures are the same as in Fig. 1. Secondary shifts >0.15 ppm are highlighted purple. Residues experiencing large chemical shift changes were plotted onto the CP crystal structure (C). CPα is shaded blue; CPβ is shaded yellow. Residues whose chemical shift changed significantly upon V-1 binding are highlighted magenta. N and C termini of CPα and CPβ are indicated. Molecular structures were rendered using the program MolMol (64).

FIGURE 5.

Chemical shift map of CP binding on V-1. The domain arrangement of V-1 is shown (A) aligned on top the secondary structures of V-1, corresponding to the NMR structure (37) (B). Chemical shift changes were plotted against residue number for V-1. Regions affected by binding are highlighted green. Chemical shift changes in V-1 were plotted onto the ribbon diagram of V-1 (C) and its N and C termini are indicated.

FIGURE 6.

Intermolecular paramagnetic relaxation enhancement. The ratios of intensity of CP resonance peaks in the paramagnetic sample to those in the diamagnetic sample (orange bars in A and B) were plotted against CPα (A) and CPβ (B) residue number. Secondary structure elements plotted above the intensities are the same as in previous figures. Black dashed line at I_p/I_d = 0.6 indicates the upper threshold used for PRE. Residues experiencing significant PRE were highlighted orange on the CP ribbon diagram (C) where the CP subunits are colored as in Fig. 3.

FIGURE 7.

Structure of CP-V-1 complex. The backbone superposition, based on CP coordinates, of the 10 lowest energy structures of the complex of V-1/CP is shown in A. The CPαβ ribbon is shaded gray, and the V-1 backbone is shaded green. The orientation is the same as in B. The ribbon diagram of the lowest energy structure of the V-1/CP complex is shown in B. The location of the PRE center on V-1 at position 7 is shown as a red sphere. V-1 is shaded green, and CP coloring is the same as in Fig. 3. N and C termini are indicated for V-1 (green) and CP (black).

FIGURE 8.

Characterization of the CP/V-1 interaction surface. Charged residues in the CP basic patch and residues on V-1 thought to interact in the binding surface were labeled on the surface of CPαβ (A) and V-1 (B). Each surface is colored based on charge, with negatively charged residues in red; positively charged residues in blue. An enlargement of the V-1/CP binding interface is shown in C. Residues on CPα, CPβ, and V-1 are labeled orange, yellow, and white, respectively. V-1 is represented as a charged surface, and CP is represented as a ribbon with the same coloring as in previous figures (C). Residues on CP thought to be involved in electrostatic interactions with V-1 are shown as sticks, and the label coloring corresponds to that in A and B.

FIGURE 9.

CP mutation analysis and mechanism of CP sequestering. V-1 binding to CP mutants was assayed by fluorescence anisotropy of V-1 in A and B. Data points represent the average value of three experiments, and error bars represent the average S.D. CP mutants are indicated according to color. Mutations that had no effect on V-1 binding are as follows: CPα1β2(K181E) (blue), CPα1β2(L258K,L262K,V263K,A265K,L266K) (orange), and wild-type CPα1β2 (black) are shown in A. Mutations that attenuated V-1 binding, CPα1(R259E,R260E,Q261E)β2 (purple), CPα1β2(K142E,K143E) (green), and wild-type CP (black) are shown in B. Equilibrium dissociation constants are presented in Table 2. Binding between CP mutants and actin barbed ends was directly assayed by monitoring pyrene-actin fluorescence in the presence of various concentrations of CP (C and D). The CP mutants used in C and D are the same as in A and B, respectively, and the coloring is the same. The residues on CP implicated in binding V-1 (E) or actin (F), based on chemical shift mapping, or mutational analysis and cryo-EM studies (16), respectively, are highlighted. The binding site for V-1 coincides that for actin in the competitive interaction surface.