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Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array : evidence for a second binding site on monomeric actin.

Title data

Mannherz, Hans Georg ; Ballweber, Edda ; Galla, Marco ; Villard, Sylvie ; Granier, Claude ; Steegborn, Clemens ; Schmidtmann, Anja ; Jaquet, Kornelia ; Pope, Brian ; Weeds, Alan G.:
Mapping the ADF/cofilin binding site on monomeric actin by competitive cross-linking and peptide array : evidence for a second binding site on monomeric actin.
In: Journal of Molecular Biology. Vol. 366 (23 February 2007) Issue 3 . - pp. 745-755.
ISSN 0022-2836
DOI: https://doi.org/10.1016/j.jmb.2006.11.100

Abstract in another language

The binding sites for actin depolymerising factor (ADF) and cofilin on G-actin have been mapped by competitive chemical cross-linking using deoxyribonuclease I (DNase I), gelsolin segment 1 (G1), thymosin beta4 (Tbeta4), and vitamin D-binding protein (DbP). To reduce ADF/cofilin induced actin oligomerisation we used ADP-ribosylated actin. Both vitamin D-binding protein and thymosin beta4 inhibit binding by ADF or cofilin, while cofilin or ADF and DNase I bind simultaneously. Competition was observed between ADF or cofilin and G1, supporting the hypothesis that cofilin preferentially binds in the cleft between sub-domains 1 and 3, similar to or overlapping the binding site of G1. Because the affinity of G1 is much higher than that of ADF or cofilin, even at a 20-fold excess of the latter, the complexes contained predominantly G1. Nevertheless, cross-linking studies using actin:G1 complexes and ADF or cofilin showed the presence of low concentrations of ternary complexes containing both ADF or cofilin and G1. Thus, even with monomeric actin, it is shown for the first time that binding sites for both G1 and ADF or cofilin can be occupied simultaneously, confirming the existence of two separate binding sites. Employing a peptide array with overlapping sequences of actin overlaid by cofilin, we have identified five sequence stretches of actin able to bind cofilin. These sequences are located within the regions of F-actin predicted to bind cofilin in the model derived from image reconstructions of electron microscopical images of cofilin-decorated filaments. Three of the peptides map to the cleft region between sub-domains 1 and 3 of the upper actin along the two-start long-pitch helix, while the other two are in the DNase I loop corresponding to the site of the lower actin in the helix. In the absence of any crystal structures of ADF or cofilin in complex with actin, these studies provide further information about the binding sites on F-actin for these important actin regulatory proteins.

Further data

Item Type: Article in a journal
Refereed: No
Additional notes: PubMed-ID: 17196218
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry > Chair Biochemistry - Univ.-Prof. Dr. Clemens Steegborn
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry
Result of work at the UBT: No
DDC Subjects: 500 Science > 540 Chemistry
Date Deposited: 14 Apr 2015 10:36
Last Modified: 14 Apr 2015 10:36
URI: https://eref.uni-bayreuth.de/id/eprint/10142