S-100 (α and β) binding peptide (TRTK-12) blocks S-100/GFAP interaction: identification of a putative S-100 target epitope within the head domain of GFAP

Abstract

Alignment of previously characterized S-100 (alpha and beta)-binding peptides (J. Biol. Chem. 270, 14651-14658) has enabled the identification of a putative S-100 target epitope within the head domain of glial fibrillary acidic protein (GFAP). The capacity of a known peptide inhibitor of S-100 protein (TRTK-12), homologous to this region, to perturb the interaction of S-100 (alpha and beta) and GFAP (J. Biol. Chem 268, 12669-12674) was investigated. Fluorescence spectrophotometry and chemical cross-linking analyses determined TRTK-12 to disrupt S-100:GFAP interaction in a dose- and Ca(2+_dependent manner. TRTK-12 also inhibited S-100's ability to block GFAP assembly and to mediate disassembly of preformed glial filaments. Each of these events was strictly dependent upon the presence of calcium and inhibitory peptide, maximal inhibition occurring at a concentration of TRTK-12 equivalent to the molar amount of S-100 monomer present. Together with our recent report demonstrating TRTK-12 also blocks the interaction of S-100 protein with the actin capping protein, CapZ, these results suggest TRTK-12 functions as a pleiotropic inhibitor of S-100 function. Availability of a functional inhibitor of S-100 will assist the further characterization of S-100 protein function in vitro and in vivo. Moreover, this report provides additional evidence supportive of a role for S-100 as a multi-faceted regulator of cytoskeletal integrity.

Specificity and Zn2+ Enhancement of the S100B Binding Epitope TRTK-12

“…It is intriguing that secondary structure prediction methods show that TRTK-12 should form an amphipathic ␣-helix between residues Lys 4 and Leu 11 . If this structure exists in the S100B-protein complex, it would place residues Thr , and Ile 11 on one side of the helix, allowing integral contact of Leu 10 and Ile 11 with S100B. On the surface, this would provide differences between the interactions of TRTK-12 with Ca 2ϩ -S100B compared with p53.…”

“…These studies showed that a 12-residue sequence containing the consensus motif (K/R)(L/ I)XWXXIL was sufficient to bind to S100B in a calcium-sensitive manner. Further studies have shown this peptide (TRTK-12) successfully competes with other proteins such as glial fibrillary acidic protein and CapZ for calcium-sensitive S100B binding (10,18). Similar approaches have identified several 15-residue sequences, analogous to the sequences for myosin light chain kinase, melittin, and mastoparan, as representative samples of the calcium-dependent target proteins for calmodulin (19).…”

Novel Interaction of the Dopamine D2Receptor and the Ca2+Binding Protein S100B: Role in D2Receptor Function

“…CapZ for calcium-sensitive S100B binding (Ivanenkov et al, 1995;Bianchi et al, 1996). This consensus sequence for S100B binding has been identified in 25 proteins found to interact in vitro with S100B (McClintock and Shaw, 2000).…”

“…A putative S100B-binding motif is located near the N terminus of the third intracellular loop of the D 2 receptor. The amino acid sequence for the rat D 2 receptor was analyzed based on the previous reports (Ivanenkov et al, 1995;Bianchi et al, 1996;McClintock and Shaw, 2000). A putative S100B binding motif close to the N terminus (amino acids: 233-240) of the third intracellular loop of the D 2 receptor is aligned with TRTK-12 peptide characterized by Ivanenkov et al (1995).…”

The calcium-modulated proteins, S100A1 and S100B, as potential regulators of the dynamics of type III intermediate filaments

“…Several lines of evidence suggest that S100B and S100A1 interact with the Nterminal head of both GFAP and desmin, thereby sequestering unassembled subunits and blocking the head-to-tail association of subunits into IF polymers (53,(56)(57)(58) (Figure 3). 1) Neither headless GFAP or desmin nor headless/tailless GFAP or desmin (i.e., their rod domain) as obtained by enzymatic cleavage was able to interact with either S100 protein.…”