Identification of contact sites in the actin-thymosin beta 4 complex by distance-dependent thiol cross-linking.

Binding sites of actin and thymosin beta 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 Cys10, Cys374, or the sulfur atom of the ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S), the actin derivatives were reacted with synthetic thymosin beta 4 analogs containing a cysteine at one of the positions 6, 17, 28, 34, and 40. Immediate cross-linking as followed by UV spectroscopy was found for Cys374 of actin and Cys6 of thymosin beta 4, indicating that the N terminus of thymosin beta 4 is in close proximity (< or = 9.2 A) to the C terminus of actin. In contrast, only insignificant reactivity was measured for all thymosin beta 4 analogs when the cross-linkers were anchored at Cys10 of actin. A second contact site was identified by cross-linking of Cys17 and Cys28 in thymosin beta 4 with the ATP gamma S derivative bound to actin, indicating that the hexamotif of thymosin beta 4 (positions 17-22) is in close proximity (< or = 9.2 A) to the nucleotide. The importance of the amino acids 17 and 28 in thymosin beta 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.