Identification of Effector Binding Sites on S100β: Studies with Guanylate Cyclase and p80, a Retinal Phosphoprotein

S100 beta is a calcium-binding protein, which regulates the activities of several enzymes and inhibits the phosphorylation of a variety of protein kinase C substrates in a calcium-dependent manner. The protein was recently found to activate a retinal membrane guanylate cyclase, and in this paper, we report that it inhibits the phosphorylation of an 80 kDa retinal protein (p80). Structurally, S100 beta consists of two EF-hands connected by a hinge region. In view of its small size, wide distribution in a variety of tissues, and regulation of many different proteins, it is of interest to identify the sites on the protein that interact with the effectors, and to determine if the same sites are responsible for interaction with different effectors. We addressed these questions with the use of synthetic peptides with sequences corresponding to different regions of S100 beta and testing their effects on the protein's activation of guanylate cyclase, and inhibition of p80 phosphorylation. Peptides with sequences corresponding to effector interaction sites were anticipated to either block or simulate the effects of S100 beta. The results show that two regions of S100 beta interact with effectors: the C-terminal region of Thr81-Glu91 and the hinge region of Leu32-Leu40. The synthetic peptide containing the latter sequence blocked the S100 beta activation of guanylate cyclase and inhibition of p80 phosphorylation, while the peptide containing the former sequence blocked cyclase activation and simulated S100 beta in inhibiting p80 phosphorylation. By determining the effects of including or excluding dithiothreitol in the assays, we observed that the cysteine residue in the C-terminal region of S100 beta (Cys84) participates in the regulation of guanylate cyclase but not of p80 phosphorylation. We conclude from these results that the C-terminal and hinge regions of S100 beta are important in the regulation of effector proteins and that Cys84 is essential for interaction with only specific effectors.