Disruption of the utrophin-actin interaction by monoclonal antibodies and prediction of an actin-binding surface of utrophin

Monoclonal antibody (mAb) binding sites in the N-terminal actin-binding dom ain of utrophin have been identified using phage-displayed peptide librarie s, and the mAbs have been used to probe functional regions of utrophin invo lved in actin binding, mAbs were characterized for their ability to interac t with the utrophin actin-binding domain and to affect actin binding to utr ophin in sedimentation assays. One of these antibodies was able to inhibit utrophin-F-actin binding and was shown to recognize a predicted helical reg ion at residues 13-22 of utrophin, close to a previously predicted actin-bi nding site: Two other mAbs which did not affect actin binding recognized pr edicted loops in the second calponin homology domain of the utrophin actin- binding domain. Using the known three-dimensional structure of the homologo us actin-binding domain of fimbrin, these results have enabled us to determ ine the likely orientation of the utrophin actin-binding domain with respec t to the actin filament.