Characterization of S-100b Binding Epitopes. IDENTIFICATION OF A NOVEL TARGET, THE ACTIN CAPPING PROTEIN, CapZ

Abstract

Short amino acid sequences that interact with the Ca2+ binding protein S-100b were identified by screening a bacteriophage random peptide display library. S-100b binding bacteriophages were selected by Ca2+-dependent affinity chromatography, and the sequence of the random peptide insert contained in 51 clones was determined. Alignment of the sequence of 44 unique S-100b binding peptides identified a common motif of eight amino acids. A subgroup of peptides that contained sequences with the highest degree of similarity had the consensus motif (K/R)(L/I)XWXXIL, 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 identified a similar sequence in the COOH-terminal region of the α-subunit of actin capping proteins. The peptide TRTKIDWNKILS (TRTK-12), corresponding to the region of greatest homology within this region of the subunit of actin capping proteins (e.g. amino acids 265-276 in CapZα1 and CapZα2), was synthesized and shown by fluorescence spectrophotometry to bind S-100b in a Ca2+-dependent manner. Gel overlay and cross-linking experiments demonstrated the interaction of S-100b with CapZ to be Ca2+ dependent. Moreover, this interaction was blocked by addition of TRTK-12 peptide. These results identify Ca2+-dependent S-100b target sequence epitopes and designate the carboxyl terminus of the α-subunit of actin capping proteins, like CapZ, to be a target of S-100b activity. The high level of conservation within this region of actin capping proteins and the apparent high affinity of this interaction strongly suggest that the interaction between S-100b and the α-subunit of actin capping proteins is biologically significant. Short amino acid sequences that interact with the Ca2+ binding protein S-100b were identified by screening a bacteriophage random peptide display library. S-100b binding bacteriophages were selected by Ca2+-dependent affinity chromatography, and the sequence of the random peptide insert contained in 51 clones was determined. Alignment of the sequence of 44 unique S-100b binding peptides identified a common motif of eight amino acids. A subgroup of peptides that contained sequences with the highest degree of similarity had the consensus motif (K/R)(L/I)XWXXIL, 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 identified a similar sequence in the COOH-terminal region of the α-subunit of actin capping proteins. The peptide TRTKIDWNKILS (TRTK-12), corresponding to the region of greatest homology within this region of the subunit of actin capping proteins (e.g. amino acids 265-276 in CapZα1 and CapZα2), was synthesized and shown by fluorescence spectrophotometry to bind S-100b in a Ca2+-dependent manner. Gel overlay and cross-linking experiments demonstrated the interaction of S-100b with CapZ to be Ca2+ dependent. Moreover, this interaction was blocked by addition of TRTK-12 peptide. These results identify Ca2+-dependent S-100b target sequence epitopes and designate the carboxyl terminus of the α-subunit of actin capping proteins, like CapZ, to be a target of S-100b activity. The high level of conservation within this region of actin capping proteins and the apparent high affinity of this interaction strongly suggest that the interaction between S-100b and the α-subunit of actin capping proteins is biologically significant.