Identification of Contact Sites in the Actin-Thymosin β4 Complex by Distance-dependent Thiol Cross-linking

Abstract

Binding sites of actin and thymosin ␤4 were investigated using a set of bifunctional thiol-specific reagents, which allowed the insertion of cross-linkers of defined lengths between cysteine residues of the complexed proteins. After the cross-linkers were attached to actin specifically at either Cys 10 , Cys 374 , or to the sulfur atom of the ATP analog adenosine 5-O-(thiotriphosphate) (ATP␥S), the actin derivatives were reacted with synthetic thymosin ␤4 analogs containing a cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate crosslinking as followed by UV spectroscopy was found for Cys 374 of actin and Cys 6 of thymosin ␤4, indicating that the N terminus of thymosin ␤4 is in close proximity ( 9.2 Å) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin ␤4 analogs when the cross-linkers were anchored at Cys 10 of actin. A second contact site was identified by cross-linking of Cys 17 and Cys 28 in thymosin ␤4 with the ATP␥S derivative bound to actin, indicating that the hexamotif of thymosin ␤4 (positions 17-22) is in close proximity ( 9.2 Å) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin ␤4 for the interaction with actin was emphasized by the finding that thymosin analogs containing cysteine in these positions exhibited strongly reduced abilities to inhibit actin polymerization.The physiological conditions within nonmuscle cells favor the assembly of actin monomers. Therefore, the pool of monomeric actin has to be maintained by complexation with small G-actin binding proteins. Particularly thymosin ␤4 (T␤4) 1 , which forms a 1:1 complex with actin monomers, is believed to be involved in preventing actin polymerization (1-6). Dissociation constants of the actin⅐T␤4 complex were found to be in the range of 0.4 -0.7 M for platelet actin and 0.7-2.0 M for muscle actin (4, 7). While complexed with T␤4, the exchange of the bound nucleotide in actin is retarded (8). In a detailed study, Vancompernolle et al. (9) have shown that binding of T␤4 to actin is mainly mediated by the hexamotif LKKTET (17-22), since loss of this sequence is paralleled by an almost complete loss of inhibitory activity. Alterations in the N-terminal part (1-16) of the peptide strongly influence the inhibitory activity of T␤4, whereas alterations in the C-terminal part (31-43) seem to be of minor importance (9). As shown by 1 H NMR spectroscopy (10, 11) T␤4 does not contain an ordered conformation in aqueous solution but tends to form an ␣-helical conformation between residues 5 and 16 (11). It has been proposed that T␤4 is likely to adopt a unique conformation upon binding actin (12).One of the binding sites of T␤4 on the actin molecule seems to be located in subdomain 1 as suggested by cross-linking studies (13,14). In order to gain more knowledge about contact sites in the actin⅐T␤4 complex, we performed a structural analysis using bifunctional thiol-specific reagents of the type alkylene-bis-[5-dithio-(2-nitrobenzoic acid)] for intermolecular crosslinki...

The ternary complex of DNase I, actin and thymosin β4

“…This was confirmed by the observation that the increase in absorbance was paralleled by a corresponding increase of a 47 kDa band in SDS-PAGE, representing the conjugate of the actin derivative (42 kDa) and the TIM analog (5 kDa) [12]. In the present study a similar 47 kDa band was found (see below), indicating that even in the presence of DNase I the crosslinking reaction had proceeded specifically.…”

“…The sole exposed thiol group of actin (cysteine 374) was selectively modified, via a disulfide bridge, with a 9.2 ,A linker bearing a thiol-capturing moiety (reaction 1). In a previous study [12] we demonstrated that the resulting actin derivative binds TIM, as well as the analog Cys6TIM, with an affinity comparable to that shown by actin itself [12]. On the addition of Cys6TIM, complex formation leads to a high-yield crosslinking reaction, which can be followed spectrophotometrically from the release of a stoichiometric amount of 2-nitro-5-thiobenzoate (reaction 2).…”

“…Since an X-ray analysis of the actin-TiM complex has so far not been achieved we have tried to identify contact sites between the two proteins by a chemical approach [12]. For this, five TIM analogs were synthesized, each of them with one cysteine residue substituted for a hydrophobic amino acid in the TIM chain.…”

“…Thymosin [~4 was isolated from bovine lungs according to Spangelo et al [17], the thymosin 154 analog (S-isopropylthio)-L-Cys6T[34 was prepared according to [12]. DNase I was purchased from Sigma (Mtmchen).…”

“…The relatively low affinity of T~4 for actin (ca. 1 ~tM [4]) may be functionally significant in allowing the instant release of polymerizable actin into cytoplasm, if required.Since an X-ray analysis of the actin-TiM complex has so far not been achieved we have tried to identify contact sites between the two proteins by a chemical approach [12]. For this, five TIM analogs were synthesized, each of them with one cysteine residue substituted for a hydrophobic amino acid in the TIM chain.…”