Daisy Dewitte

The actin binding site of thymosin beta 4 mapped by mutational analysis.

We characterized in detail the actin binding site of the small actin‐sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full‐length T beta 4 variants. The N‐terminal part (residues 1–16) and a hexapeptide motif (residues 17–22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross‐linking, complex formation in native gels and actin‐sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N‐terminal part needs to adopt an alpha‐helix for actin binding and interacts through a patch of hydrophobic residues (6M‐I‐F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N‐terminal alpha‐helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta‐thymosin family and in addition to this we identify a similar pattern in the C‐terminal headpiece of villin and dematin.

Mutational analysis of human profilin I reveals a second PI(4,5)-P2 binding site neighbouring the poly(L-proline) binding site

BackgroundProfilin is a small cytoskeletal protein which interacts with actin, proline-rich proteins and phosphatidylinositol 4,5-bisphosphate (PI(4,5)-P2). Crystallography, NMR and mutagenesis of vertebrate profilins have revealed the amino acid residues that are responsible for the interactions with actin and poly(L-proline) peptides. Although Arg88 of human profilin I was shown to be involved in PI(4,5)-P2-binding, it was suggested that carboxy terminal basic residues may be involved as well.ResultsUsing site directed mutagenesis we have refined the PI(4,5)-P2 binding site of human profilin I. For each mutant we assessed the stability and studied the interactions with actin, a proline-rich peptide and PI(4,5)-P2 micelles. We identified at least two PI(4,5)-P2-binding regions in human profilin I. As expected, one region comprises Arg88 and overlaps with the actin binding site. The second region involves Arg136 in the carboxy terminal helix and neighbours the poly(L-proline) binding site. In addition, we show that adding a small protein tag to the carboxy terminus of profilin strongly reduces binding to poly(L-proline), suggesting local conformational changes of the carboxy terminal α-helix may have dramatic effects on ligand binding.ConclusionsThe involvement of the two terminal α-helices of profilin in ligand binding imposes important structural constraints upon the functions of this region. Our data suggest a model in which the competitive interactions between PI(4,5)-P2 and actin and PI(4,5)-P2 and poly(L-proline) regulate profilin functions.

Analogous F-actin Binding by Cofilin and Gelsolin Segment 2 Substantiates Their Structural Relationship

Cofilin is representative for a family of low molecular weight actin filament binding and depolymerizing proteins. Recently the three-dimensional structure of yeast cofilin and of the cofilin homologs destrin and actophorin were resolved, and a striking similarity to segments of gelsolin and related proteins was observed (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagaka, F. (1996) Cell 85, 1047–1055; Fedorov, A. A., Lappalainen, P., Fedorov, E. V., Drubin, D. G., and Almo, S. C. (1997) Nat. Struct. Biol. 4, 366–369; Leonard, S. A., Gittis, A. G., Petrella, E. C., Pollard, T. D., and Lattman, E. E. (1997) Nat. Struct. Biol. 4, 369–373). Using peptide mimetics, we show that the actin binding site stretches over the entire cofilin α-helix 112–128. In addition, we demonstrate that cofilin and its actin binding peptide compete with gelsolin segments 2–3 for binding to actin filaments. Based on these competition data, we propose that cofilin and segment 2 of gelsolin use a common structural topology to bind to actin and probably share a similar target site on the filament. This adds a functional dimension to their reported structural homology, and this F-actin binding mode provides a basis to further enlighten the effect of members of the cofilin family on actin filament dynamics.

Unbalancing the phosphatidylinositol-4,5-bisphosphate-cofilin interaction impairs cell steering.

Cofilin is a key player in actin dynamics during cell migration. Its activity is regulated by (de)phosphorylation, pH, and binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)]. Here, we here use a human cofilin-1 (D122K) mutant with increased binding affinity for PI(4,5)P(2) and slower release from the plasma membrane to study the role of the PI(4,5)P(2)-cofilin interaction in migrating cells. In fibroblasts in a background of endogenous cofilin, D122K cofilin expression negatively affects cell turning frequency. In carcinoma cells with down-regulated endogenous cofilin, D122K cofilin neither rescues the drastic morphological defects nor restores the effects in cell turning capacity, unlike what has been reported for wild-type cofilin. In cofilin knockdown cells, D122K cofilin expression promotes outgrowth of an existing lamellipod in response to epidermal growth factor (EGF) but does not result in initiation of new lamellipodia. This indicates that, next to phospho- and pH regulation, the normal release kinetics of cofilin from PI(4,5)P(2) is crucial as a local activation switch for lamellipodia initiation and as a signal for migrating cells to change direction in response to external stimuli. Our results demonstrate that the PI(4,5)P(2) regulatory mechanism, that is governed by EGF-dependent phospholipase C activation, is a determinant for the spatial and temporal control of cofilin activation required for lamellipodia initiation.

A phage display technique for a fast, sensitive, and systematic investigation of protein-protein interactions.

Phage display is a technique in which a foreign protein or peptide is presented at the surface of a (filamentous) bacteriophage. This system, developed by Smith [(1985), Science228, 1315–1317], was originally used to create large libraries of antibodies for the purpose of selecting those that strongly bound a particular antigen. More recently it was also employed to present peptides, domains of proteins, or intact proteins at the surface of phages, again to identify high-affinity interactions with ligands. Here we want to illustrate the use of phage display, in combination with PCR saturation mutagenesis, for the study of protein–protein interactions. Rather than selecting for mutants having high affinity, we systematically investigate the binding of every variant with its natural ligand. Via a modified ELISA we can calculate a relative affinity. As a model system we chose to display thymosin β4 on the phage surface in order to study its interaction with actin.

Evidence for an actin binding helix in gelsolin segment 2; have homologous sequences in segments 1 and 2 of gelsolin evolved to divergent actin binding functions?

Gelsolin is built up of six homologous segments that perform different functions on actin. Segments 1 and 2, which are suggested to be highly similar in their overall folds, bind monomeric and filamentous actin respectively. A long α‐helix in segment 1 forms the major contact site of this segment with actin. We show that sequence 197–226 of segment 2, equivalent to the region around the actin binding helix in segment 1, contains F‐actin binding activity. Consequently, positionally similar parts of segment 1 and 2 are implicated in the actin contact and solvent exposed residues in these parts must have evolved differentially to meet their different actin binding properties.