Interaction of formin FH2 with skeletal muscle actin. EPR and DSC studies

Abstract

Formins are highly conserved proteins that are essential in the formation and regulation of the actin cytoskeleton. The formin homology 2 (FH2) domain is responsible for actin binding and acts as an important nucleating factor in eukaryotic cells. In this work EPR and DSC were used to investigate the properties of the mDia1-FH2 formin fragment and its interaction with actin. MDia1-FH2 was labeled with a maleimide spin probe (MSL). EPR results suggested that the MSL was attached to a single SH group in the FH2. In DSC and temperature-dependent EPR experiments we observed that mDia1-FH2 has a flexible structure and observed a major temperature-induced conformational change at 41 °C. The results also confirmed the previous observation obtained by fluorescence methods that formin binding can destabilize the structure of actin filaments. In the EPR experiments the intermolecular connection between the monomers of formin dimers proved to be flexible. Considering the complex molecular mechanisms underlying the cellular roles of formins this internal flexibility of the dimers is probably important for manifestation of their biological functions.

Fig. 1

Rate of polymerization of actin by unlabelled and MSL–formin, and formin dialyzed in different buffers: (a) actin without formin; (b) actin with unlabelled formin; (c) actin with formin dialyzed in DTT-containing buffer T; (d) actin and formin dialyzed in DTT-free buffer T; (e) actin and MSL-labeled formin dialyzed in DTT-free buffer T; (f) actin and NEM-labeled formin dialyzed in DTT-containing buffer T; (g) actin and NEM-labeled formin not dialyzed. The protein concentrations were: 5 μM for actin and 1 μM for formin FH2 in all cases. Actin was stored in buffer A (0.2 mM ATP, 0.1 mM CaCl₂, 4 mM Tris–HCl, pH 8.0) and labeled with pyrene (5 %). Polymerization of magnesium–actin was started by addition of 1 mM MgCl₂ and 50 mM KCl

Fig. 2

Upper 2 spectra: conventional EPR spectra of MSL–formin and its complex with F-actin. Lower 2 spectra: conventional EPR spectra of MSL–G-actin and MSL–F-actin. In contrast with MSL–F-actin, the spectra of MSL–formin contain two components with different rotational mobility. The peak heights of the low-field components are labeled I ₊₁ and I _+1m

Fig. 3

(a) Temperature dependence of 2${\text{A}}_{\text{zz}}^{\prime }$ of MSL–formin as a function of temperature. At approximately 41 °C a breakpoint is apparent. (b) Temperature dependence of the hyperfine splitting constants of: MSL–formin complex with F-actin (1:5 mol/mol) (filled squares), MSL–F-actin complex with formin (5:1 mol/mol) (asterisks), and MSL–F-actin without formin (filled triangles)

Fig. 4

Comparison of DSC traces of formin samples: formin, G-actin, formin plus F-actin (1:3 mol/mol), and F-actin. The heat flows are plotted in arbitrary units to demonstrate the differences between the transition temperatures and the peak width at half maximum of the protein samples

Fig. 5

Van’t Hoff plot for the double integral ratio of the first two components in the spectrum of MSL–formin. From the slope of the straight line the free energy change (ΔG) was determined to be 6.7 kJ mol⁻¹ K⁻¹

Fig. 6

Change of the natural logarithm of the rotational correlation time for the MSL–formin complex with F-actin (1:5 mol/mol) as a function of temperature. Note the change of the activation energy after the breakpoint

Fig. 7

Plots of I ₊₁/I _+1m against reciprocal absolute temperature. Filled triangles, MSL–formin; squares, MSL–formin complex with F-actin (1:10 mol/mol); circles, MSL–formin complex with F-actin (1:5 mol/mol)