Polymerisation of chemically cross-linked actin:thymosin β 4 complex to filamentous actin: alteration in helical parameters and visualisation of thymosin β 4 binding on F-actin 1 1Edited by W. Baumeister

Ballweber, Edda; Hannappel, Ewald; Huff, Thomas; Stephan, Harald; Haener, Markus; Taschner, Nicole; Stoffler, Daniel; Aebi, Ueli; Mannherz, Hans Georg

doi:10.1006/jmbi.2001.5281

Cited by 53 publications

(55 citation statements)

References 40 publications

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“…This suggests that the villin headpiece faces actin close to this residue at the barbed end of the actin molecule and that the biotin-neutravidin moiety sterically hinders binding. Thymosin ␤4 is also proposed to contact the barbed end (5), but a model presented recently shows a location of thymosin ␤4 shifted away from the barbed end (44). Given the observed competition between thymosin ␤4 and the villin headpiece (15), our results suggest that the binding sites only partly overlap.…”

Section: Discussionmentioning

confidence: 55%

A Phage Display-based Method for Determination of Relative Affinities of Mutants

Rossenu

Leyman²,

Dewitte

et al. 2003

Journal of Biological Chemistry

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We propose phage display combined with enzymelinked immunosorbent assay as a tool for the systematic analysis of protein-protein interactions by investigating the binding behavior of variants to a partner protein. Via enzyme-linked immunosorbent assay we determine both the amount of fusion protein presented at the phage surface and the amount of complex formed, the ratio of which is proportional to the affinity. Hence this method enables us to calculate the relative affinities of a large number of mutants. As model systems, we investigated actin-binding motifs conserved in a number of proteins binding monomeric or filamentous actin. The hexapeptide motifs LKKTET, present in thymosin ␤4, and LKKEKG, present in the villin headpiece, were mutated, and the variants were analyzed. Study of the positional tolerance allows postulating that the motifs, although similar in primary structures adopt different conformations when bound to actin. In addition, our data show that the second and the fourth amino acid of the thymosin ␤4 motif and the first three residues of the villin headpiece motif are most important for actin binding. The latter result challenges the charged crown hypothesis for the villin headpiece filamentous actin interaction.As a consequence of various genome-sequencing projects, novel proteins are being identified, many of which may take part in new interactions. Efforts are going on to tackle the discovery of protein-protein interactions globally (1), but there is also need for novel methods for the analysis of these interactions, preferably easy, reliable, and high through-put techniques. Several means to probe amino acids that contribute to the binding of two proteins have been developed. X-ray crystallography and NMR may yield superior information about the interface of proteins at atomic resolution (2, 3). However, both methods are intrinsically difficult (even for noncomplexed proteins) and time-consuming and require expensive and sophisticated equipment, as well as large amounts of biological material. Cross-linking of interacting proteins followed by mass spectroscopy or conventional sequence determination of covalently coupled peptide fragments has limited application (4 -6). Probing the accessible surface in protein complexes by deuterium exchange has also been employed (7). But by far the two most popular methods are the use of peptide mimetics either in solution (8) or on membranes (9, 10) and especially site-directed mutagenesis (11, 12). However, in the latter powerful technique to study functions of proteins or interactions between proteins, one often faces the difficulty of choosing the position and type of amino acid exchange to be introduced. To circumvent this, one may perform a saturation mutagenesis at several positions thought to be important for the interaction. Obviously this creates a new problem, i.e. screening a large number of mutants in a systematic way.We propose to combine saturation mutagenesis, phage display and ELISA 1 for the systematic screening and quantification of pr...

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Section: Discussionmentioning

confidence: 55%

A Phage Display-based Method for Determination of Relative Affinities of Mutants

Rossenu

Leyman²,

Dewitte

et al. 2003

Journal of Biological Chemistry

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“…The first structural confirmation that the extended structure of Tb4 is also maintained upon binding to actin was obtained by reconstruction of electron microscopical images from filaments formed from chemically cross-linked actin:Tb4 complex, Fig. 1C [Ballweber et al, 2002]. This reconstruction indicated that the N-and C-terminal helixes formed by residues 5 to 15 and 30 and 40 were attached to the regions of the actin molecule formed by subdomains 1 and 3 and subdomains 2 and 4, respectively.…”

Section: Structure Of the Actin:thymosin B4 Complexmentioning

confidence: 81%

“…Both exhibit important intracellular functions and are present outside and inside of cells in high concentrations. Adenine nucleotides are released from cells under [Ballweber et al, 2002]. (D,E) give the 3D-structures of the actin complexes with the D1 subunit of ciboulot [Hertzog et al, 2004], (E) with the fusion protein G1 and the C-terminal half of Tb4 [Irobi et al, 2004].…”

Section: Extracellular Activities Of the B-thymosinsmentioning

confidence: 99%

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

Mannherz

Hannappel

2009

Cell Motil. Cytoskeleton

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108

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The b-thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin b4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin b4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response. Various paracrine effects have indeed been reported for these peptides such as cardiac protection, angiogenesis, stimulation of wound healing, and hair growth. Besides these paracrine effects, it was noted that b-thymosins occur in high concentration in the cytoplasm of many eukaryotic cells and bind to the cytoskeletal component actin. Subsequently it became apparent from in vitro experiments that they preferentially bind to monomeric (G-)actin and stabilize it in its monomeric form. Due to this ability the b-thymosins are the main intracellular actin sequestering factor, i.e., they posses the ability to remove monomeric actin from the dynamic assembly and disassembly processes of the actin cytoskeleton that constantly occur in activated cells. In this review we will concentrate on the intracellular activity and localization of the b-thymosins, i.e., their modulating effect on the actin cytoskeleton. Cell Motil. Cytoskeleton 66: 839-851, 2009. '

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“…Thymosin␤4 can also be cross-linked to F-actin, albeit only at very high concentrations (Carlier et al, 1996). Hence, F-actin-binding by this module is not unprecedented (see also Ballweber et al, 2002). Note that, in the nonsequestering, F-actin-binding and thus functionally most divergent, repeat 3, the hexapeptide motif is identical to the one of thymosin ␤4, whereas in repeat 4, the pure sequestering module, the motif is more diverse.…”

Section: Discussionmentioning

confidence: 98%

TetraThymosinβ Is Required for Actin Dynamics inCaenorhabditis elegansand Acts via Functionally Different Actin-binding Repeats

Troys

Ono

Dewitte

et al. 2004

MBoC

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Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosin␤ expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosin␤ has four repeats, each related to the actin monomer-sequestering protein thymosin␤4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosin␤ binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G-and F-actin interaction sites, tetraThymosin␤ may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosin␤ is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosin␤ is of crucial importance at specific developmental stages requiring actin polymerization.

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Polymerisation of chemically cross-linked actin:thymosin β 4 complex to filamentous actin: alteration in helical parameters and visualisation of thymosin β 4 binding on F-actin 1 1Edited by W. Baumeister

Cited by 53 publications

References 40 publications

A Phage Display-based Method for Determination of Relative Affinities of Mutants

A Phage Display-based Method for Determination of Relative Affinities of Mutants

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

TetraThymosinβ Is Required for Actin Dynamics inCaenorhabditis elegansand Acts via Functionally Different Actin-binding Repeats

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