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 Cys(10), Cys(374), or to 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 crosslinking as followed by UV spectroscopy was found for Cys(374) of actin and Cys(6) of thymosin beta 4, indicating that the N terminus of thymosin beta 4 is in close proximity (less than or equal to 9.2 Angstrom) 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 Cys(10) of actin. A second contact site was identified by cross-linking of Cys(17)and Cys(28) 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 (less than or equal to 9.2 Angstrom) 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.