Journal of Molecular Biology
Volume 315, Issue 4, 25 January 2002, Pages 613-625
Journal home page for Journal of Molecular Biology

Regular article
Polymerisation of chemically cross-linked actin:thymosin β4 complex to filamentous actin: alteration in helical parameters and visualisation of thymosin β4 binding on F-actin1

https://doi.org/10.1006/jmbi.2001.5281Get rights and content

Abstract

The β-thymosins are intracellular monomeric (G-)actin sequestering proteins forming 1:1 complexes with G-actin. Here, we analysed the interaction of thymosin β4 with F-actin. Thymosin β4 at 200 μM was chemically cross-linked to F-actin. In the presence of phalloidin, the chemically cross-linked actin:thymosin β4 complex was incorporated into F-actin. These mixed filaments were of normal appearance when inspected by conventional transmission electron microscopy after negative staining. We purified the chemically cross-linked actin:thymosin β4 complex, which polymerised only when phalloidin and the gelsolin:2-actin complex were present simultaneously. Using scanning transmission electron microscopy, the mass-per-length of control and actin:thymosin β4 filaments was found to be 16.0(±0.8) kDa/nm and 18.0(±0.9) kDa/nm, respectively, indicating an increase in subunit mass of 5.4 kDa. Analysis of the helical parameters revealed an increase of the crossover spacing of the two right-handed long-pitch helical strands from 36.0 to 40.5 nm. Difference map analysis of 3-D helical reconstruction of control and actin:thymosin β4 filaments yielded an elongated extra mass. Qualitatively, the overall size and shape of the difference mass were compatible with published data of the atomic structure of thymosin β4. The deduced binding sites of thymosin β4 to actin were in agreement with those identified previously. However, parts of the difference map might represent subtle conformational changes of both proteins occurring upon complex formation.

Introduction

Among the numerous actin-binding proteins there is a limited number of proteins that interact preferentially with monomeric (G-)actin. These proteins have the capacity to depolymerise filamentous (F-)actin, stabilise it in its monomeric form and/or to sequester it from the equilibrium between G- and F-actin, thus preventing its incorporation into the polymer moiety. A prototype of an actin-sequestering protein is thymosin β4 (Tβ4) and related peptides (the β-thymosins), which in some cells are present in high enough concentration to stabilize about 50 % of the intracellular actin in G-form.1, 2, 3, 4 The β-thymosins have a molecular mass of roughly 5 kDa and are found in almost every eukaryotic cell. They form a 1:1 complex with G-actin of intermediate binding affinity (KD = 0.2 to 5 μM).

Very little is known about the binding site of thymosin β4 on actin. Attempts have been made to identify residues or regions on actin involved in thymosin β4 binding by chemical cross-linking using 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide (EDC)5, 6 or by competition of thymosins with other actin-binding proteins..7, 8, 9 Both approaches have led to the proposal that thymosin β4 binds to the N-terminus located on subdomain 1 and to Glu167 of subdomain 3 of actin.6, 8 It has been suggested that thymosin β4 might exist as an elongated molecule that stretches from the bottom of subdomain 3 to the DNase I binding region on subdomain 2 of actin.6

The β-thymosins are able to intracellularly shift the G/F-equilibrium of actin towards the monomeric state by removing (sequestering) G-actin from this equilibrium. It has been proposed that a rapid increase in the number of free ends of F-actin upon cell stimulation leads to the incorporation of free monomeric actin into F-actin. Indeed, in vitro experiments have shown that an increase in the number of free actin filament ends results in dissociation of the actin:thymosin β4 complex in order to maintain the equilibrium between free G-actin and the actin:thymosin β4 complex.10

Within the framework of this model, thymosin β4 is able to bind to only G-actin. However, it has been demonstrated recently that Tβ4 is able to interact with filamentous actin,11, 12 since it was shown that addition of high concentrations of thymosin β4 (>200 μM) to polymerising actin decreased the critical concentration of free actin to polymerise, indicating the participation of the actin:Tβ4 complex in the polymerisation reaction. Similarly, it has been reported that over-expression of thymosin β4 in fibroblasts induces increased formation of actin containing stress fibres and, at the same time, decreased the amount of monomeric actin.12

We therefore analysed the interaction of thymosin β4 with preformed F-actin, and the ability of the actin:thymosin β4 complex chemically cross-linked by EDC to copolymerise with native actin or to form actin filaments by itself. Our data demonstrated that in the simultaneous presence of phalloidin and gelsolin, the purified cross-linked actin:thymosin β4 complex can be induced to polymerise into filamentous actin. Structural analysis of electron micrographs of negatively stained actin:Tβ4 filaments revealed a 4.5 nm increase of the crossover spacing of the two long-pitch helical strands. Difference map analysis of a 3-D helical reconstruction of control and actin:thymosin β4 filaments revealed the possible contact sites of thymosin β4 with actin, and suggested the occurrence of subtle conformational changes in actin upon thymosin β4 binding.

Section snippets

Thymosin β4 can be cross-linked to F-actin

It has been shown that thymosin β4 can be cross-linked to F-actin.11 However, since kinetic analysis demonstrated that F-actin depolymerising ability by free thymosin β4 is most probably due to a shift in the G/F-equilibrium and the formation of actin:Tβ4,11 we repeated these experiments in the absence or presence of phalloidin, which stabilises preformed F-actin in the presence of Tβ4.10 After incubation overnight (about 16 hours) of F-actin and thymosin β4 added at 200 μM (14-fold excess over

Discussion

Kinetic experiments analysing the effect of high concentrations of thymosin β4 on the Cc or the G/F-equilibrium had demonstrated that thymosin β4 was able to interact with filamentous actin.11, 12 Therefore, we analysed the ability of thymosin β4 to interact with filamentous actin and of chemically cross-linked actin:thymosin β4 complex to incorporate into or to even form actin polymers by itself. Using chemical cross-linking, we showed that thymosin β4 was able to bind to F-actin and when

Materials

The chemical cross-linker 1-ethyl-3[3-(dimethylamino)propyl]carbodiimide (EDC) was obtained from Pierce (Rockford, IL, USA). Monodansylcadaverine, tissue type transglutaminase (guinea pig liver) and subtilisin were commercial products of Sigma Corp., München, Germany. All other reagents were of analytical grade.

Protein preparations

Rabbit skeletal muscle actin was purified as described23 as modified by Ballweber7 and taken up in G-buffer (5 mM Hepes-OH (pH 7.4), 0.1 mM CaCl2, 0.5 mM NaN3, 0.2 mM ATP). Human plasma

Acknowledgements

It is a pleasure for us to thank Mrs Ulrike Ritenberg for expert technical assistance and Dr T. Holak (Martinsried, Germany) for supplying the coordinates of thymosin β4; the Deutsche Forschungsgemeinschaft, the Swiss National Science Foundation, the M.E. Müller Foundation of Switzerland, and the Canton Basel-Stadt for financial support.

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