CHARACTERIZATION OF S-100B BINDING EPITOPES - IDENTIFICATION OF A NOVEL TARGET, THE ACTIN CAPPING PROTEIN, CAPZ

Short amino acid sequences that interact with the Ca2+ binding protein S-100b were identified by screening a bacteriophage random peptide di splay library. S-100b binding bacteriophages were selected by Ca2+-dep endent affinity chromatography, and the sequence of the random peptide insert contained in 51 clones was determined. Alignment of the sequen ce of 44 unique S-100b binding peptides identified a common motif of e ight amino acids. A subgroup of peptides that contained sequences with the highest degree of similarity had the consensus motif (K/R)(L/I)XW XXIL, in which predominantly P, S, and N were found in position 3, and S and D were found in position 5. Analysis of sequence databanks iden tified a similar sequence in the COOH-terminal region of the alpha-sub unit of actin capping proteins. The peptide TRTKIDWNKILS (TRTK-12), co rresponding to the region of greatest homology within this region of t he subunit of actin capping proteins (e.g. amino acids 265-276 in CapZ alpha 1 and CapZ alpha 2), was synthesized and shown by fluorescence spectrophotometry to bind S-100b in a Ca2+-dependent manner. Gel overl ay and cross-linking experiments demonstrated the interaction of S-100 b with CapZ to be Ca2+ dependent. Moreover, this interaction was block ed by addition of TRTK-12 peptide. These results identify Ca2+-depende nt S-100b target sequence epitopes and designate the carboxyl terminus of the alpha-subunit of actin capping proteins, like CapZ, to be a ta rget of S-100b activity. The high level of conservation within this re gion of actin capping proteins and the apparent high affinity of this interaction strongly suggest that the interaction between S-100b and t he alpha-subunit of actin capping proteins is biologically significant .