Inhibitor peptide SNP-1 binds to a soluble form of BST-1/CD157 at a 2:2 stoichiometry

Abstract

Recently we have identified a 15-mer peptide, SNP-1, by a random phage library that can bind to bone marrow stromal cell antigen-1 (BST-1)/CD157 [Sato, A., Yamamoto, S., Ishihara, K., Hirano, T. and Jingami, H. (1999) Biochem. J. 337, 491-496]. SNP-1 inhibits BST-1 ADP-ribosyl cyclase activity uncompetitively with a K(i) value of 180 ± 40 nM. In this study we analysed biophysically the SNP-1 binding to a soluble form of BST-1 (sBST-1). Equilibrium binding data of wild-type SNP-1 from surface plasmon resonance studies gave a K(d) value of 500 ± 35 nM. Titration calorimetry analysis showed that the binding reaction is exothermic at 20 °C. The values of K(d) = 211 nM, enthalpy change, ΔH = -18.68 kcal · mol-1, and saturated molar ratio of bound SNP-1 per sBST-1, N = 0.8 mol · mol-1 were obtained. On the basis of the molecular masses of SNP-1 and sBST-1 calculated by analytical ultracentrifugation, the stoichiometry of the binding was determined to be 2 : 2. Electron microscopy also revealed the dimer form of sBST-1. To delineate the core residue of SNP-1 responsible for binding, each amino acid residue has been replaced by alanine. A region from amino acid residues 7-12 appeared to be critical for the SNP-1 binding to sBST-1. The substitution of the first residue, His, to Ala led to a reduction in binding, suggesting that the N- terminal residue is also crucial.