Formins Regulate Actin Filament Flexibility through Long Range Allosteric Interactions

Abstract

The members of the formin family nucleate actin polymerization and play essential roles in the regulation of the actin cytoskeleton during a wide range of cellular and developmental processes. In the present work, we describe the effects of mDia1-FH2 on the conformation of actin filaments by using a temperature-dependent fluorescence resonance energy transfer method. Our results revealed that actin filaments were more flexible in the presence than in the absence of formin. The effect strongly depends on the mDia1-FH2 concentration in a way that indicates that more than one mechanism is responsible for the formin effect. In accordance with the more flexible filament structure, the thermal stability of actin decreased and the rate of phosphate dissociation from actin filaments increased in the presence of formin. The interpretation of the results supports a model in which formin binding to barbed ends makes filaments more flexible through long range allosteric interactions, whereas binding of formin to the sides of the filaments stabilizes the protomer-protomer interactions. These results suggest that formins can regulate the conformation of actin filaments and may thus also modulate the affinity of actin-binding proteins to filaments nucleated/capped by formins.Formins are evolutionarily conserved proteins (1, 2) that activate signaling pathways and nucleate actin filaments independently of the Arp2/3 complex (3-6). In mammalian cells, formins play a role in the formation of stress fibers, cell motility, signaling, gene transcription, and embryonic development (7-13). In yeast, formins organize cytoplasmic actin cables and the contractile ring (1, 3, 14 -17). Formins are composed of multiple domains (2), which can include formin homology domains (18) (FH1, FH2, FH3), N-terminal GTPase-binding domain (GBD), 3 and C-terminal diaphanous-autoregulatory domain (DAD). FH1 and FH2 domains are present in all formins (15). The proline-rich FH1 can interact with profilin, with factors involving the SH3 domain and the Src family kinases (9,14,17,19,20). The FH2 domain is required for the interaction with actin, for the stabilization of microtubules, and for serum response factor activation (5, 9, 12, 21). Diaphanous-related formins involve GBD and DAD domains (22). In some diaphanousrelated formins, binding of activated Rho relieves intramolecular interactions between the DAD and N-terminal sequences (19,23).Biophysical characterization of formin fragments from mammalian sources (from mouse, mDia1 (4, 24 -26) and mDia3 (25)), from Saccharomyces cerevisiae (Bni1p and Bnr1p) (3, 24), and from Schizosaccharomyces pombe (Cdc12p) (27) established that they were potent actin nucleators in vitro and that the FH2 domain was essential for the nucleation. Recent structural studies have given insights into the molecular mechanisms responsible for the formin functions. The structures of the FH2 domains from mDia1 (25), from Bni1p (28), from the complex of actin with Bni1-FH2 (29), and from the complex of the GBD from mDia1 and Rho A (3...

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

“…These observations made in the absence of formins are in good agreements with the results of the classical Stern-Volmer analyses (Fig. 2B) and also with the data from previous quenching studies on actin filaments [18,36,37].…”

“…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.…”

“…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].…”