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β-Thymosin (βT) and WH2 domains are widespread, intrinsically disordered actin-binding peptides that display significant sequence variability and different regulations of actin self-assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin-β4, or enhance motility by directing polarized filament assembly like Ciboulot βT. We combined mutational, functional or structural analysis by X-ray crystallography, SAXS (small angle X-ray scattering) and NMR on Thymosin-β4, Ciboulot, TetraThymosinβ and the long WH2 domain of WASP-interacting protein. The latter sequesters G-actin with the same molecular mechanisms as Thymosin-β4. Functionally different βT/WH2 domains differ by distinct dynamics of their C-terminal half interactions with G-actin pointed face. These C-terminal interaction dynamics are controlled by the strength of electrostatic interactions with G-actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G-actin sequestration in both isolated long βT and WH2 domains. The results open perspectives for elucidating the functions of βT/WH2 domains in other modular proteins.

The beta-thymosin/WH2 actin-binding module shows an amazing adaptation to multifunctionality. The beta-thymosins are genuine G-actin sequesterers of moderate affinity for G-actin, allowing an efficient regulation of the G-actin/F-actin ratio in cells by amplifying changes in the critical concentration for filament assembly. In contrast, the first beta-thymosin domain of the protein Ciboulot makes with G-actin a complex that supports filament growth, such as profilin-actin. We illustrate how the use of engineered chimeric proteins, actin-binding and polymerization assays, crystallographic, NMR, and SAXS structural approaches complement each other to decipher the molecular basis for the functional versatility of these intrinsically disordered domains when they form various 1:1 complexes with G-actin. Multifunctionality is expanded in tandem repeats of WH2 domains present in WASP family proteins and proteins involved in axis patterning like Cordon-Bleu and Spire. The tandem repeats generate new functions such as filament nucleation and severing, as well as barbed end binding, which add up to the G-actin sequestering activity. Novel regulation pathways in actin assembly emerge from these additional activities.

Cell-penetrating peptides can cross cell membranes and are commonly seen as biologically inert molecules. However, we found that some cell-penetrating peptides could remodel actin cytoskeleton in oncogene-transformed NIH3T3/EWS-Fli cells. These cells have profound actin disorganization related to their tumoral transformation. These arginine- and/or tryptophan-rich peptides could cross cell membrane and induce stress fiber formation in these malignant cells, whereas they had no perceptible effect in non-tumoral fibroblasts. In addition, motility (migration speed, random motility coefficient, wound healing) of the tumor cells could be decreased by the cell-permeant peptides. Although the peptides differently influenced actin polymerization in vitro, they could directly bind monomeric actin as determined by NMR and calorimetry studies. Therefore, cell-penetrating peptides might interact with intracellular protein partners, such as actin. In addition, the fact that they could reverse the tumoral phenotype is of interest for therapeutic purposes.

beta-thymosins are acknowledged G-actin sequesterers. However, in the recent years, the conserved beta-thymosins/WH2 actin-binding module, has been identified in a large number of proteins that all interact with actin and play diverse functions in cell motility. The functional evolution of the WH2 domain has been approached by a combination of structural and biochemical methods, using thymosin beta4 (Tbeta4) and Ciboulot, a 3 beta-thymosin repeat protein from Drosophila as models. Ciboulot binds actin like Tbeta4 but promotes actin assembly like profilin. The first repeat of Ciboulot (D1) has the profilin function of the whole protein. The crystal structure of Ciboulot-actin shows that the major interaction with G-actin lies in the N-terminal amphipathic helix of D1. By point mutagenesis the sequestering activity of Tbeta4 can be changed into a profilin activity. ((1)H, (15)N)-NMR studies show that the functional switch from inhibition to promotion of actin assembly is linked to a change in the dynamics of interaction of the central and C-terminal regions of the WH2 domain with subdomains 1 and 2 of G-actin. Further systematic mutagenesis studies have been performed by engineering a series of chimeras of Ciboulot and Tbeta4. Proteins displaying either profilin function or enhanced sequestering activity compared to Tbeta4 have been characterized. The results provide insight into the structural basis for the regulation of the multiple functions of the WH2 domain.

Thymosin beta4 is a major actin-sequestering protein, yet the structural basis for its biological function is still unknown. This study provides insight regarding the way this 43-amino acid peptide, mostly unstructured in solution, binds to monomeric actin and prevents its assembly in filaments. We show here that the whole backbone of thymosin beta4 is highly affected upon binding to G-actin. The assignment of all amide protons and nitrogens of thymosin in the bound state, obtained using a combination of NMR experiments and selective labelings, shows that thymosin folds completely upon binding and displays a central extended region flanked by two N- and C-terminal helices. The cleavage of actin by subtilisin in the DNase I binding loop does not modify the structure of thymosin beta4 in the complex, showing that the backbone of the peptide is not in close proximity to segment 42-47 of actin. The combination of our NMR results and previously published mutation and cross-link data allows a better characterization of the binding mode of thymosins on G-actin.

The widespread beta-thymosin/WH2 actin binding domain has versatile regulatory properties in actin dynamics and motility. beta-thymosins (isolated WH2 domain) maintain monomeric actin in a "sequestered" nonpolymerizable form. In contrast, when repeated in tandem or inserted in modular proteins, the beta-thymosin/WH2 domain promotes actin assembly at filament barbed ends, like profilin. The structural basis for these opposite functions is addressed using ciboulot, a three beta-thymosin repeat protein. Only the first repeat binds actin and possesses the function of ciboulot. The region that shows the strongest interaction with actin is an amphipathic N-terminal alpha helix, present in all beta-thymosin/WH2 domains, which recognizes the ATP bound actin structure and uses the shear motion of actin linked to ATP hydrolysis to control polymerization. Crystallographic ((1)H, (15)N), NMR, and mutagenetic data reveal that the weaker interaction of the C-terminal region of beta-thymosin/WH2 domain with actin accounts for the switch in function from inhibition to promotion of actin assembly.