THYMOSIN BETA(4) BINDS ACTIN IN AN EXTENDED CONFORMATION AND CONTACTSBOTH THE BARBED AND POINTED ENDS

The beta-thymosins are a family of highly polar peptides which serve i n vivo to maintain a reservoir of unpolymerized actin monomers. In vit ro, beta-thymosins form 1:1 complexes with actin monomers and inhibit both polymerization and exchange of the bound nucleotide. Circular dic hroism data indicate that free thymosin beta(4) is predominantly unstr uctured, containing at most six residues of alpha-helix, and that up t o 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 residue s in actin and thymosin beta(4) were identified by zero-length cross-l inking followed by isolation and sequencing of cross-linked peptides. After carbodiimide-mediated cross-linking, Lys-3 of thymosin beta(4) w as cross-linked to Glu-167 of actin, and Lys-18 of thymosin beta(4) wa s 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 ex-helical region o f thymosin beta 4 and place it on the barbed end; thymosin beta(4) can thus block actin polymerization sterically. After transglutaminase-me diated cross-linking, Lys-38 of thymosin beta(4) was cross-linked to G ln-41 of actin, placing the C-terminal region of thymosin beta 4 in co ntact with subdomain 2 on the pointed end; thymosin beta 4 may sterica lly block actin polymerization at the pointed end as well as the barbe d 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.