Thymosin β₄ Binds Actin in an Extended Conformation and Contacts both the Barbed and Pointed Ends

Abstract

The beta-thymosins are a family of highly polar peptides which serve in vivo to maintain a reservoir of unpolymerized actin monomers. In vitro, beta-thymosins form 1:1 complexes with actin monomers and inhibit both polymerization and exchange of the bound nucleotide. Circular dichroism data indicate that free thymosin beta 4 is predominantly unstructured, containing at most six residues of alpha-helix, and that up to six additional residues may adopt an alpha-helical conformation upon binding actin. NMR data indicate that many parts of thymosin beta 4 are not in tight contact with actin. Contacts between specific residues in actin and thymosin beta 4 were identified by zero-length cross-linking followed by isolation and sequencing of cross-linked peptides. After carbodiimide-mediated cross-linking, Lys-3 of thymosin beta 4 was cross-linked to Glu-167 of actin, and Lys-18 of thymosin beta 4 was cross-linked to one of the the N-terminal acidic residues of actin (Asp-1-Glu-4); the cross-linked actin residues lie within subdomains 3 and 1, respectively. These two contacts flank the alpha-helical region of thymosin beta 4 and place it on the barbed end; thymosin beta 4 can thus block actin polymerization sterically. After transglutaminase-mediated cross-linking, Lys-38 of thymosin beta 4 was cross-linked to Gln-41 of actin, placing the C-terminal region of thymosin beta 4 in contact with subdomain 2 on the pointed end; thymosin beta 4 may sterically block actin polymerization at the pointed end as well as the barbed end of the monomer. The distance between the pointed-end and barbed-end contacts requires that the C-terminal half of thymosin beta 4 be in a predominantly extended conformation.

Formation and Implications of a Ternary Complex of Profilin, Thymosin β4, and Actin

“…These results imply that, in agreement with results of Safer et al (12) and De La Cruz et al (13), the C terminus of thymosin ␤ 4 likely contributes to the binding of the intact protein by direct interactions with actin. It is unlikely that the 5 residues N-terminal to our peptide are responsible for binding of intact thymosin ␤ 4 to actin in the presence of the peptide because others have shown that these residues are not important in this regard (9).…”

“…Also, this assumption has been used as the basis for the analysis of data concerning putative functions of profilin other than monomer sequestration (5,6,11). However, the recent evidence that the surface of interaction between thymosin ␤ 4 and actin is extensive, involving both subdomains 1 and 2 of actin (12,13), implies that there is no structural basis for competitive binding between these actinbinding proteins, and has lead us to re-investigate the hypothesis that profilin and thymosin ␤ 4 could form a ternary complex with actin. Additional support for this hypothesis is provided by data showing that some variations in the C terminus of thymosin ␤ 4 alter actin affinity and explain, in part, the differences in actin binding activity of ␤-thymosin isoforms (14,15).…”

TetraThymosinβ Is Required for Actin Dynamics inCaenorhabditis elegansand Acts via Functionally Different Actin-binding Repeats

“…(C) Same experiment as in A and B, but using tetraThymosin␤ mutant tT␤R 1,2,4 that is mers simultaneously, most probably via repeats 1, 2, and 4. Similar to thymosin␤4 (Safer et al, 1997), tetraThymosin␤ likely adopts an extended conformation (it is largely unstructured as a free protein, our unpublished results) enabling the formation of complexes with varying stoichiometry. Analogously to our results, actobindin is shown to contain two functional actin-binding modules (Vancompernolle et al, 1991) and to complex an actin dimer (Bubb et al, 1994).…”

Prolyl Oligopeptidase Is Involved in Release of the Antifibrotic Peptide Ac-SDKP

“…10 Tissue was desegregated and diluted up to 150 g/mL total protein with cold phosphate-saline buffer; 100-L aliquots of the homogenate were incubated at 37°C with: (1) Safer, University of Pennsylvania, Philadelphia, Pa), 20 was dissolved in water. Three different POPi were used: Z-prolyl-prolinal (Z-ProPro, Biomol), Fmoc-prolyl-pyrrolidine-2-nitrile (Fmoc-Pro-PyrrCN, a gift of S. Wilk, Mount Sinai School of Medicine, NY), 21 and S17092 (a gift of P. Vanhoutte, Institut de Recherches Internationales Servier, France).…”