Dual roles of Gln137 of actin revealed by recombinant human cardiac muscle alpha-actin mutants

Abstract

The actin filament is quite dynamic in the cell. To determine the relationship between the structure and the dynamic properties of the actin filament, experiments using actin mutants are indispensable. We focused on Gln(137) to understand the relationships between two activities: the conformational changes relevant to the G- to F-actin transition and the activation of actin ATPase upon actin polymerization. To elucidate the function of Gln(137) in these activities, we characterized Gln(137) mutants of human cardiac muscle alpha-actin. Although all of the single mutants, Q137E, Q137K, Q137P, and Q137A, as well as the wild type were expressed by a baculovirus-based system, only Q137A and the wild type were purified to high homogeneity. The CD spectrum of Q137A was similar to that of the wild type, and Q137A showed the typical morphology of negatively stained Q137A F-actin images. However, Q137A had an extremely low critical concentration for polymerization. Furthermore, we found that Q137A polymerized 4-fold faster, cleaved the gamma-phosphate group of bound ATP 4-fold slower, and depolymerized 5-fold slower, as compared with the wild-type rates. These results suggest that Gln(137) plays dual roles in actin polymerization, in both the conformational transition of the actin molecule and the mechanism of ATP hydrolysis.

FIGURE 1.

Location of the mutated residue Gln¹³⁷ in actin. A, overall crystal structure of ATP-G-actin (Protein Data Bank code 1YAG). Gln¹³⁷ and ATP are indicated by space-filling and ball-and-stick representations, respectively. Subdomains 1-4 are indicated by the corresponding number. B, side view image of the colored region in A. The Mg²⁺ and the water molecule at the position suitable for an in-line attack to the γ-phosphate group of the bound ATP are indicated. These images were created with MOLSCRIPT (39, 40).

FIGURE 2.

SDS-PAGE pattern of purified recombinant actins. Purified WT and Q137A are shown in the middle and right lanes, respectively. In the left lane are molecular mass standards in kDa.

FIGURE 3.

CD spectra and thermal stability of G-actin. A, CD spectra of G-actins measured at 25 and 70 °C (solid and dashed curves, respectively). B, thermal melting profiles of G-actins monitored by the ellipticity at 222 nm, at temperatures increasing from 25 to 70 °C at a constant rate of 1 °C/min. Recombinant (thick curves, WT, black; Q137A, gray) and tissue-purified actins (thin curves, skeletal muscle, black; cardiac muscle, gray) are shown. Samples contained 5 μm actin, 5 mm Tris-HCl, pH 8.0, 0.1 mm CaCl₂, 0.25 mm ATP, and 0.5 mm DTT. Each profile was obtained after averaging the measurements of at least two independent preparations. T_m values are shown in Table 1.

FIGURE 4.

Electron micrographs of negatively stained F-actin. The polymerized WT and Q137A at the early steady state of polymerization were negatively stained with uranyl acetate. Samples contained 12.5 μm actin, 100 mm KCl, 2 mm MgCl₂, 0.5 mm ATP, and 33 mm imidazole-HCl, pH 7.4, in G-buffer. Electron micrographs were recorded at a magnification of 40,000. The thick particle in the right panel is a tobacco mosaic virus particle, which was included to make the staining homogeneous. Crossover repeats of F-actins are shown in Table 2.

FIGURE 5.

Actin polymerization kinetics. A and B, time courses of the light scattering of WT, Q137A, and tissue-purified actins (skeletal muscle, black; cardiac muscle, gray) were followed after the initiation of polymerization. The solution for the polymerization measurements contained 25 μm actin, 100 mm KCl, 2 mm MgCl₂, and 0.5 mm ATP in G-buffer, at 25 °C. The light scattering to 90° was measured at 660 nm. The time courses indicated were obtained by averaging four measurements each for WT and skeletal muscle actin, and three measurements each for Q137A and cardiac muscle actin. For each actin species, at least three independent preparations were used. C, maximal rate of apparent elongation. The maximal rate was determined by using the time course of light scattering measured in A and B. Bars indicate ± S.D. D, relationship between the maximal rates of apparent elongation and the concentration of actin. The time courses of light scattering were measured at actin concentrations of 5, 12.5, and 25 μm and are shown in supplemental Fig. S1. The maximal rates of apparent elongation are plotted versus the actin concentrations in a double logarithmic plot. WT (○), Q137A (▵), skeletal muscle (□), and cardiac muscle (□) actin are shown.

FIGURE 6.

Actin ATPase rate during polymerization. A and B, time courses of P_i release from WT, Q137A, and tissue-purified actins (skeletal muscle, black; cardiac muscle, gray) during polymerization. The concentrations of P_i released from actin solutions at 25 μm were measured with an EnzChek phosphate assay kit. The solution conditions for polymerization were the same as those in the experiments described in Fig. 5. The indicated time courses were obtained by averaging three measurements for WT and tissue-purified actins and four measurements for Q137A. For each actin species, at least two independent preparations were used. C, concentrations of P_i released and total ADP at 45 min after initiation of polymerization. The actin concentration was 25 μm. The concentrations of P_i released correspond to those at the ends of the curves in A and B. The total ADP concentration was measured by reverse-phase HPLC and averaged over six measurements for WT and cardiac muscle actin, four for Q137A, and eight for skeletal muscle actin. For each actin species, at least two independent preparations were used. Bars indicate ± S.D.

FIGURE 7.

Relationships between the time courses of polymerization and P_i release. A, skeletal muscle actin is shown in black, and cardiac muscle actin is shown in gray; B, WT; C, Q137A. In each panel, the time course of light scattering (continuous curve) shown in Fig. 5, A and B, and the time course of P_i release (dotted curve) shown in Fig. 6, A and B, are superposed. The units of the vertical axis for the light scattering time course were converted to the concentration of F-actin.

FIGURE 8.

Time course of apparent F-actin depolymerization. Depolymerization was initiated by the addition of 7.5 μm vitamin D-binding protein into a solution containing 5 μm F-actin at the early steady state of polymerization, and the reaction was incubated on ice. The concentration of F-actin was determined by densitometry of the pellet fraction obtained by ultracentrifugation. [F-actin]₀ and [F-actin]_t indicate the concentration of F-actin before and at time t after the initiation of depolymerization, respectively. Each data point was obtained after averaging five measurements for WT (•), Q137A (▴), and cardiac muscle actin (□), or three measurements for skeletal muscle actin (•). Bars indicate ± S.D.