Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor
Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor
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DOI：
10.1074/jbc.271.30.18277
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发表时间：
1996-07-26
影响因子：
4.8
通讯作者：
Mikoshiba, K
中科院分区：
文献类型：
--
作者：
Yoshikawa, F;Morita, M;Mikoshiba, K
为了确定I型三磷酸肌醇受体(IP(3)R1)与三磷酸肌醇(IP3)结合的结构决定因素，我们建立了一种在大肠杆菌中表达IP(3)R1(T734)N端734个氨基酸的方法，其中包含IP3结合区。在大肠杆菌中表达的T734蛋白与来自小鼠小脑的天然IP(3)R具有相似的结合IP3的特异性和亲和力。在缺失突变中，T734从N端到215位残基连续缺失，显著降低了IP3结合活性。然而，当C末端略有缺失(对220、223和225位残基)时，结合活性被恢复，在前228个氨基酸上进一步的N末端缺失再次完全取消了它。C端579个残基的缺失不影响结合活性，568个残基的C端缺失完全取消了结合活性。此外，表达的356个氨基酸的多肽(残基224-579)具有特异的结合活性。综上所述，226-578残基足够并足够接近特定IP3结合的最小区域，从而形成了IP3结合的“核心”。对T734基因N端650个氨基酸中的41个碱性Arg和Lys残基进行定点突变。我们发现，IP(3)R家族中广泛分布并在所有成员中保守的10个残基的单一氨基酸替换显著降低了结合活性。其中3个(Arg-265、Lys-508和Arg-511)对特异性结合是关键的，而Arg-568与多种肌醇磷酸盐的结合专一性有关。我们认为这10个残基中的一些形成了一个基本的口袋，与IP3的带负电荷的磷酸基团相互作用。
To define the structural determinants for inositol 1,4,5-trisphosphate (IP3) binding of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1), we developed a means of expressing the N-terminal 734 amino acids of IP(3)R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP(3)R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved, Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity. Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding ''core.'' Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP(3)R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3.