The effects of ADF/cofilin and profilin on the conformation of the ATP-binding cleft of monomeric actin

Abstract

Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer's ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound epsilon-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound epsilon-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound epsilon-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.

Figure 1

Frequency domain measurement of the ɛ-ATP labeled actin monomers. (Upper panel) The difference between the measured data values and the fits (residuals) were plotted for the phase delay (solid symbols) and modulation ratio (shaded symbols) at different modulation frequencies. (Lower panel) The change of the phase delay (solid symbols) and the modulation ratio (shaded symbols) of the signal form ɛ-ATP labeled actin monomers was recorded in a frequency range between 2 and 64 MHz in the absence of acrylamide.

Figure 2

Results from the steady-state quenching measurements with ɛ-ATP labeled actin monomers. (A) The change of the fluorescence spectrum of the ɛ-ATP labeled monomers (5 μM) in the presence of increasing acrylamide concentration (0–0.3 M). (B) The Stern-Volmer plot of three independent steady-state quenching experiments with the actin-bound ɛ-ATP (5 μM actin) in the presence of different acrylamide concentrations (0–0.3M). Solid lines represents the fits obtained with the Eq. 4. The calculated K_{SV_S} value is 0.24 ± 0.05 M⁻¹ for the actin bound ɛ-ATP molecules.

Figure 3

Quenching of the fluorescence lifetime of ɛ-ATP labeled actin monomers with acrylamide. (A) The upper panel shows the longer lifetime component (τ₂) that belongs to the actin bound ɛ-ATP whereas the lower panel shows the quenching of the shorter lifetime (τ₁) of the free ɛ-ATP. (B) The Stern-Volmer plot of the shorter lifetime of the free ɛ-ATP (τ₁) in the presence of different acrylamide concentrations (0–0.3M). The solid line represents the fit of the Eq. 2 to the obtained experimental data. The value for the K_{SV_D} was 49.20 ± 4.28 M⁻¹ for the free ɛ-ATP in the solution. The error bars represent the values of SD.

Figure 4

Quenching of the fluorescence of ɛ-ATP labeled actin monomers with acrylamide in the presence of ADF/cofilin molecules. (A) The change of the fluorescence spectrum of the ɛ-ATP labeled monomers (5 μM) in the presence of ADF/cofilin (15 μM) with increasing acrylamide concentration (0–0.3 M). (B) The Stern-Volmer plots from the steady-state quenching experiments with 5 μM ɛ-ATP labeled actin monomers in the presence of 15 μM cofilin (○) at different acrylamide concentrations (0–0.3 M). The calculated K_{SV_S} value was 0.034 ± 0.017 M⁻¹ for the actin bound ɛ-ATP in the presence of cofilin.

Figure 5

Quenching of the fluorescence of ɛ-ATP labeled actin monomers with acrylamide in the presence of profilin. (A) The change of the fluorescence spectrum of the ɛ-ATP labeled monomers (5 μM) in the presence of profilin (20 μM) with increasing acrylamide concentration (0–0.3 M). (B) The Stern-Volmer plots from the steady-state quenching experiments with 5 μM ɛ-ATP labeled actin monomers in the presence of 20 μM profilin (□) at different acrylamide concentrations (0–0.3 M). The calculated K_{SV_S} value for the actin bound ɛ-ATP was 3.5 ± 1.5 M⁻¹ in the presence of the profilin.