THE ACTIN-BINDING SITE OF THYMOSIN BETA-4 MAPPED BY MUTATIONAL ANALYSIS

We characterized in detail the actin binding site of the small actin-s equestering protein thymosin beta 4 (T beta 4) using chemically synthe sized full-length T beta 4 variants, The N-terminal part (residues 1-1 6) and a hexapeptide motif(residues 17-22) form separate structural en tities, In both, we identified charged and hydrophobic residues that p articipate in the actin interaction using chemical cross-linking, comp lex formation in native gels and actin-sequestering experiments, Quant itative data on the activity of the variants and circular dichroism ex periments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patc h of hydrophobic residues ((6)M-I-F-12). On one side of this helix, Al so, electrostatic contacts between actin and lysine residues 18, in th e motif, and 14, in the N-terminal alpha-helix, appear important for b inding, The residues critical for contacting actin are conserved throu ghout the beta-thymosin family and in addition to this we identify a s imilar pattern in the C-terminal headpiece of villin and dematin.