Abstract
The hydrolysis of ATP accompanying actin polymerization destabilizes the filament, controls actin assembly dynamics in motile processes, and allows the specific binding of regulatory proteins to ATP- or ADP-actin. However, the relationship between the structural changes linked to ATP hydrolysis and the functional properties of actin is not understood. Labeling of actin Cys374 by tetramethylrhodamine (TMR) has been reported to make actin non-polymerizable and enabled the crystal structures of ADP-actin and 5'-adenylyl beta,gamma-imidodiphosphate-actin to be solved. TMR-actin has also been used to solve the structure of actin in complex with the formin homology 2 domain of mammalian Dia1. To understand how the covalent modification of actin by TMR may affect the structural changes linked to ATP hydrolysis and to evaluate the functional relevance of crystal structures of TMR-actin in complex with actin-binding proteins, we have analyzed the assembly properties of TMR-actin and its interaction with regulatory proteins. We show that TMR-actin polymerized in very short filaments that were destabilized by ATP hydrolysis. The critical concentrations for assembly of TMR-actin in ATP and ADP were only an order of magnitude higher than those for unlabeled actin. The functional interactions of actin with capping proteins, formin, actin-depolymerizing factor/cofilin, and the VCA-Arp2/3 filament branching machinery were profoundly altered by TMR labeling. The data suggest that TMR labeling hinders the intramolecular movements of actin that allow its specific adaptative recognition by regulatory proteins and that determine its function in the ATP- or ADP-bound state.
Highlights
ATP destabilizes actin-actin interactions in the filament and affects the structure of actin enough to specify the recognition of ATP- and ADP-actin by different regulatory proteins, yet the nature of the structural change that supports the functional importance of this reaction in actin following ATP hydrolysis remains a debated issue
In contrast with these conclusions, uncomplexed tetramethylrhodamine (TMR)-labeled actin harbors a closed structure in both the ADP- and AMPPNP-bound states; in addition, actin subdomain 2 adopts a novel helical fold in the ADP-bound state, whereas it displays the conventional disordered fold in the AMPPNP-bound state [2, 8]. This observation suggests that the structural change in subdomain 2 is linked to ATP hydrolysis and is responsible for the weakening of longitudinal actinactin bonds in the filament. This proposed structural effect of ATP hydrolysis was questioned because the TMR probe, which locates at the shear zone linking subdomains 1 and 3 at the barbed face of actin, may interfere with the structural changes linked to the binding of ATP or ADP to unlabeled, fully functional actin [12]
We show that TMR-actin polymerizes, with a 30-fold higher critical concentration than unlabeled actin, into very short filaments that are destabilized by ATP hydrolysis
Summary
Proteins—Actin was purified from rabbit skeletal muscle and isolated in the monomeric CaATP-G-actin form by gel filtration chromatography on Superdex 300 in buffer A (5 mM Tris-Cl (pH 7.8), 1 mM dithiothreitol, 0.1 mM CaCl2, 0.2 mM ATP, and 0.01% NaN3). Different batches of labeled material gave identical values of the critical concentration and parameters of interaction with regulatory proteins. The following scheme and derived set of differential equations were used, where A is ATP-G-actin, F is the filament ends, and Aadp is ADP-G-actin: Step 1, A ϩ A 3 F (nucleation); Step 2, FϩANF (growth-depolymerization); Step 3, F N 2F (fragmentation-reannealing); Step. The following values of the rate constants corresponding to Steps 1–5 were used to obtain a satisfactory fit of experimental spontaneous assembly curves recorded over an order of magnitude span in TMR-actin concentrations. The observed fluorescence was computed as follows: F ϭ f0 ϩ (A0 Ϫ Atotal)1⁄7f(F) ϩ A1⁄7f(A) ϩ Aadp1⁄7f(Aadp), where f0 is the fluorescence signal for buffer alone, f(F), f(A), and f(Aadp) are the specific fluorescence of F-actin, ATP-G-actin, and ADP-G-actin, respectively. The first parameter to be adjusted was the one that displayed the smallest S.D
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