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

Tünde Kupi,Pál Gróf,Miklós Nyitrai,József Belágyi

doi:10.1007/s00249-013-0922-0

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00249-013-0922-0) contains supplementary material, which is available to authorized users.

Highlights

The dynamics of the actin cytoskeleton––its rapid assembly and disassembly, which is essential for many cellular functions––is regulated in vivo by a variety of actin-binding proteins (ABPs) (Hild et al 2010; Lappalainen 2007; Pantaloni et al 2001; Pollard et al 2000; Pollard and Borisy 2003)
We showed that formins can be labeled on a single cysteine residue with spin probes
Spin-labeled formin can furnish information about important aspects of the intra-molecular transitions occurring in formins and in formin–actin complexes

Summary

Introduction

The dynamics of the actin cytoskeleton––its rapid assembly and disassembly, which is essential for many cellular functions––is regulated in vivo by a variety of actin-binding proteins (ABPs) (Hild et al 2010; Lappalainen 2007; Pantaloni et al 2001; Pollard et al 2000; Pollard and Borisy 2003). Experiments using fluorescence spectroscopic and paramagnetic resonance techniques have shown that formin binds to the barbed end of actin filaments and induces a change of their flexibility (Bugyi et al 2006; Kupi et al 2009; Papp et al 2006). The binding of formins to the sides of the actin filaments is less tight and stabilizes the structure of the filaments, probably by connecting neighboring protomers (Bugyi et al 2006). The DSC transients revealed the destabilization of actin filaments by formin (Bugyi et al 2006). It is expected that the interaction between actin and formin is mutual in a sense that their binding affects the conformation of both proteins. Little is known about the conformational changes in formin accompanying the actin binding

Methods

Discussion

Conclusion