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

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Abstract
The β-thymosins are a family of highly polar peptides which serve in vivo to maintain a reservoir of unpolymerized actin monomers. In vitro, β-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 β4 is predominantly unstructured, containing at most six residues of α-helix, and that up to six additional residues may adopt an α-helical conformation upon binding actin. NMR data indicate that many parts of thymosin β4 are not in tight contact with actin. Contacts between specific residues in actin and thymosin β4 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 β4 was cross-linked to Glu-167 of actin, and Lys-18 of thymosin β4 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 α-helical region of thymosin β4 and place it on the barbed end; thymosin β4 can thus block actin polymerization sterically. After transglutaminase-mediated cross-linking, Lys-38 of thymosin β4 was cross-linked to Gln-41 of actin, placing the C-terminal region of thymosin β4 in contact with subdomain 2 on the pointed end; thymosin β4 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 β4 be in a predominantly extended conformation.