Hydrophobic residues in the C-terminal region of S100A1 are essential for target protein binding butnot for dimerization

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• 作者：Dirk Osterloh ; Vasily V. Ivanenkov ; Volker Gerke
• 关键词：Ca²⁺ signals ; IP₃ ; SH-SY5Y cells
• 刊名：Cell Calcium
• 出版年：1998
• 出版时间：August 1998
• 年：1998
• 卷：24
• 期：2
• 页码：137-151
• 全文大小：2485 K

文摘

S100 proteins are a family of small dimeric proteins characterized by two EF hand type Ca ²⁺ binding motifs which are flanked by unique N- and C-terminal regions. Although shown unequivocally in only a few cases S100 proteins are thought to function by binding to, and thereby regulating, cellular target proteins in a Ca²⁺ dependent manner. To describe for one member of the family, S100A1, structural requirements underlying target protein binding, we generated specifically mutated S1 00A1 derivatives and characterized their interaction with the a subunit of the actin capping protein CapZ shown here to represent a direct binding partner for S100A1. Chemical cross-linking, ligand blotting and fluorescence emission spectroscopy reveal that removal of, or mutations within, the sequence encompassing residues 88–90 in the unique C-terminal region of S1 00A1 interfere with binding to CapZa and to TRTK12, a synthetic CapZa peptide. The S1 00A1 sequence identified contains a cluster of three hydrophobic residues (Phe-88, Phe-89 and Trp-90) at least one of which - as revealed by qualitative phenyl Sepharose binding and hydrophobic fluorescent probe spectroscopy - is exposed on the protein surface of Ca²⁺ bound S100A1. As homologous hydrophobic residues in the closely related S100B protein were shown by NMR spectroscopy of Ca²⁺ -free S100B dimers to provide intersubunit contacts [Kilby P.M., van Eldik L.J., Roberts G.C.K. The solution structure of the bovine S100B dimer in the calcium-free state. Structure 1996; 4: 1041–1052; Drohat A.C., Amburgey J.C., Abildgaard F., Starich M.R., Baldisseri D., Weber D.J. Solution structure of rat apo-S100B (beta beta) as determined by NMR spectroscopy. Biochemistry 1996; 35: 11577–11588], we characterized the physical state of the various S100Al derivatives. Analytical gel filtration and chemical cross-linking show that dimer formation is not compromised in S100A1 mutants lacking residues 88–90 or containing specific amino acid substitutions in this sequence. Thus a cluster of hydrophobic residues in the C-terminal region of S100Al is essential for target protein binding but dispensable for dimerization, a situation possibly met in other S100 proteins as well.