Widely Distributed Residues in Thymosin β4 Are Critical for Actin Binding

Actin filament assembly and disassembly in cells are regulated by several families of proteins, which interact preferentially with actin monomers or filaments (1-3). Many regulatory proteins that bind monomers also sever, cap, or nucleate actin filaments (e.g., gelsolin or the ADF/cofilin family) or promote addition of the monomer to growing filaments (e.g., profilin). The β-thymosins make up the only known family of actin binding proteins whose sole activity under physiological conditions is to sequester actin monomers, thereby generating a pool of unpolymerized actin that can be used for rapid barbed-end elongation. The most thoroughly studied β-thymosin is Tβ4,1 which is the predominant isoform in most vertebrate species (4, 5). Cross-linking and spectroscopic studies showed that Tβ4 binds actin monomers in a predominantly extended conformation and retains considerable segmental mobility when bound (6). These early findings and later structural studies (7-10) indicate that the interface between actin and Tβ4 consists of multiple small, separate contacts, which constitute a distributed binding site, rather than a compact, solvent-excluding interface. The predominantly unfolded and extended structure of Tβ4 would allow different segments to bind sequentially. Several studies have used mutagenesis or chemical modification in investigating the contributions of specific Tβ4 residues to actin binding affinity (11-15). Initial studies focused on the six-amino acid sequence LKKTET at positions 17-22, because of its homology to a well-characterized actin-binding sequence in actobindin (15) and because mutations in this region reduce the actin affinity of Tβ4 (15). Truncation at the N- or C-terminus, mutations in the α-helical region, or oxidation of M6 (11-13, 16) weakens actin monomer binding. In addition, cross-linking studies implicated K3, K18, K38, and the C-terminus of Tβ4 in forming contacts with actin (6). However, these early studies provided only semiquantitative data and did not address the contributions of specific Tβ4 residues to binding strength and mechanism or provide data about the effects of mutations on the kinetics of complex formation and dissociation. Tβ4 binding quenches the fluorescence intensity of AEDANS conjugated to cysteine 374 of actin (17) and enhances the fluorescence of nearby tryptophan residues W330 and W355 (17, 18). These spectroscopic changes were attributed to contact between the α-helical segment of Tβ4 (residues D5-L16) and the hydrophobic pocket on the barbed end of actin monomers. The decrease in AEDANS fluorescence upon binding suggests that Tβ4 displaces the fluorophore from contact with hydrophobic side chains on actin, forcing it into a more solvent-exposed environment. In this study, we investigate the effects of mutations outside the α-helical region on the kinetics and stability of actin binding, using the signal arising from contact between the α-helical region of Tβ4 and the AEDANS fluorophore. We evaluate the contributions of hydrophobic residues outside the α-helical region (L28 and I34) and of the highly conserved proline (P4, P27, and P29) and lysine (K18 and K19) residues.