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.
Reichert
A
A
Max-Planck-Institut für medizinische Forschung, Heidelberg, Germany.
Heintz
D
D
Echner
H
H
Voelter
W
W
Faulstich
H
H
D000199:Actins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents; D003553:Cystine; D004591:Electrophoresis, Polyacrylamide Gel; D008168:Lung / Q000378:metabolism; D008433:Mathematics; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism; D008961:Models, Structural; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds; D013947:Thymosin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000099201:Thymosin beta(4)
D000199:Actins; D000255:Adenosine Triphosphate; D003432:Cross-Linking Reagents; D003553:Cystine; D008840:Microfilament Proteins; D010455:Peptides; D013438:Sulfhydryl Compounds; D013947:Thymosin; D000099201:Thymosin beta(4); C022571:adenosine 5'-O-(3-thiotriphosphate)