Molecular biology of actin binding proteins: evidence for a common structural domain in the F-actin binding sites of gelsolin and  -actinin

The β-thymosins are small monomeric (G-)actin-binding proteins of 5 kDa that are supposed to act intracellularly as actin-sequestering factors stabilizing the cytoplasmic monomeric pool of actin. The binding region of thymosin β4 was determined by analysing the binding of thymosin β4 to actin complexed with DNase I, gelsolin or gelsolin segment 1. Binding was analysed by determining the increase in the critical concentration of actin polymerization by native gel electrophoresis or chemical cross-linking. The formation of a ternary complex including thymosin β4 should indicate that the actin-binding proteins attach to different sites on actin. Competition would be indicative of binding to identical or overlapping sites on actin or of a negative co-operative linkage between the two binding sites. Competition of thymosin β4 for actin binding was observed in the presence of intact gelsolin or the N-terminal gelsolin fragment, segment 1, indicating that thymosin β4 binds to a site close to or identical with the gelsolin segment 1-binding site. The ternary complex of actin-DNase I-thymosin β4 was obtained only when using the chemically cross-linked actin-thymosin β4 complex, indicating that thymosin β4 is dissociated by the binding of DNase I to actin. It is suggested that the dissociation of thymosin β4 by DNase I binding to actin is caused by negative co-operativity between their spatially separated binding sites on actin. A similar negative co-operativity was observed between DNase I and gelsolin segment 1 binding to actin. The results therefore indicate that the respective binding sites for DNase I and segment 1 on subdomains 1 and 2 of actin are linked in a negative co-operative manner.

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The utrophin actin-binding domain binds F-actin in two different modes

The Journal of Cell Biology ◽

10.1083/jcb.200111097 ◽

2002 ◽

Vol 157 (2) ◽

pp. 243-251 ◽

Cited By ~ 63

Author(s):

Vitold E. Galkin ◽

Albina Orlova ◽

Margaret S. VanLoock ◽

Inna N. Rybakova ◽

James M. Ervasti ◽

...

Keyword(s):

Binding Sites ◽

Binding Proteins ◽

Binding Domain ◽

Actin Binding ◽

Actin Binding Proteins ◽

New Approach ◽

Calponin Homology ◽

Multiple Binding Sites ◽

Actin Binding Domain ◽

Multiple Binding

Utrophin, like its homologue dystrophin, forms a link between the actin cytoskeleton and the extracellular matrix. We have used a new method of image analysis to reconstruct actin filaments decorated with the actin-binding domain of utrophin, which contains two calponin homology domains. We find two different modes of binding, with either one or two calponin-homology (CH) domains bound per actin subunit, and these modes are also distinguishable by their very different effects on F-actin rigidity. Both modes involve an extended conformation of the CH domains, as predicted by a previous crystal structure. The separation of these two modes has been largely dependent upon the use of our new approach to reconstruction of helical filaments. When existing information about tropomyosin, myosin, actin-depolymerizing factor, and nebulin is considered, these results suggest that many actin-binding proteins may have multiple binding sites on F-actin. The cell may use the modular CH domains found in the spectrin superfamily of actin-binding proteins to bind actin in manifold ways, allowing for complexity to arise from the interactions of a relatively few simple modules with actin.

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