Conformational Changes in Actin Filaments Induced by Formin Binding to the Barbed End

Abstract

Formins bind actin filaments and play an essential role in the regulation of the actin cytoskeleton. In this work we describe details of the formin-induced conformational changes in actin filaments by fluorescence-lifetime and anisotropy-decay experiments. The results show that the binding of the formin homology 2 domain of a mammalian formin (mouse mDia1) to actin filaments resulted in a less rigid protein structure in the microenvironment of the Cys374 of actin, weakening of the interactions between neighboring actin protomers, and greater overall flexibility of the actin filaments. The formin effect is smaller at greater ionic strength. The results show that formin binding to the barbed end of actin filaments is responsible for the increase of flexibility of actin filaments. One formin dimer can affect the dynamic properties of an entire filament. Analyses of the results obtained at various formin/actin concentration ratios indicate that at least 160 actin protomers are affected by the binding of a single formin dimer to the barbed end of a filament.

The effects of formins on the conformation of subdomain 1 in actin filaments

“…These intramolecular changes suggest that the subdomain 1 of the actin protomers became more flexible after the binding of formin and this change provided the structural framework for the establishment of the broader conformational distribution. This conclusion and the observed ionic strength dependence of the formin effects are in correlation with our previous observations [17][18][19].…”

“…The formin effect depended on the formin:actin molar ratio, and also on the ionic strength of the solution. These observations provided further details regarding the formin induced conformational changes in the actin filaments and corroborated our previous results [17][18][19].…”

“…Previously the effect of formins on the conformation of actin filaments was shown to be salt concentration dependent [17,18,20]. To test whether the quenching method was sensitive to the salt dependence of the conformational changes in actin we carried out the steady-state measurements at a higher ionic strength (50 mM KCl and 1 mM MgCl 2 ) as well.…”

“…At lower formin concentrations (below approximately 500 nM) the formin is binding preferably to the high affinity (K D = 20-50 nM, [6,38]) binding-sites at the barbed end of filaments. Formin binding to the barbed ends induce long range allosteric conformational changes which make the actin filaments more flexible [17,18], and result in the observed changes of the quenching parameters. At higher salt concentrations the formin binding to the filament ends is weaker and thus the development of its conformational effect in actin requires more formin.…”

“…The effect of the formins on the structure of actin has been previously investigated in the case of FH2 domain of mouse Diaphanous-related protein 1 (mDia1-FH2) and it was found that the structure of the formin-bound filaments is significantly different from those polymerized in the absence of formins. Binding of the mDia1-FH2 to barbed ends of filaments made the structure of actin filaments more flexible, while side-binding of formins stabilised the conformation of the filaments [17,18]. Subsequent experiments using EPR methods gave further evidences regarding the formin induced changes in actin filaments [19].…”